Sterile chromatography resin and use thereof in manufacturing processes

ABSTRACT

Provided herein are methods of reducing bioburden of a chromatography resin that include exposing a container including a composition including (i) a chromatography resin and (ii) a liquid including at least on alcohol to a dose of gamma-irradiation sufficient to reduce the bioburden of the container and the chromatography resin, where the at least one alcohol are present in an amount sufficient to ameliorate the loss of binding capacity of the chromatography resin after/upon exposure to the dose of gamma-irradiation. Also provided are reduced bioburden chromatography columns including the reduced bioburden chromatography resin, compositions including a chromatography resin and a liquid including at least one alcohol, methods of performing reduced bioburden column chromatography using one of these reduced bioburden chromatography columns, and integrated, closed, and continuous processes for reduced bioburden manufacturing of a purified recombinant protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/726,043, filed Aug. 31, 2018; the entire contents of whichare herein incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of biotechnology and thebiomanufacturing of recombinant proteins.

BACKGROUND

Mammalian cells including a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. In the current environment of diverse productpipelines, biotechnology companies are increasingly driven to developinnovative solutions for highly flexible and cost-effectivemanufacturing of therapeutic protein drug substances. One of thestrategies for efficiently isolating recombinant proteins is throughprocesses that include continuous chromatography (e.g., using a closedsystem). One known limitation of continuous chromatography is thepresence of contaminating agents in the system (e.g., increasedbioburden), which results in a contaminated product, a reduction in theproduction yield, and a decrease in the flow-rate (or increase in thepressure) in the system. For example, the increased bioburden within asystem can result in the complete shut down of the system.

SUMMARY

The present invention is based, at least in part, on the discovery thatgamma-irradiation of chromatography resin reduces the binding capacityof the chromatography resin and that irradiation in the presence of atleast one alcohol can help prevent this reduction in binding capacity ofa chromatography resin caused by gamma-irradiation. In view of thisdiscovery, provided herein are methods of reducing bioburden of achromatography resin that include exposing a container including acomposition including (i) a chromatography resin and (ii) a liquidincluding at least one alcohol to a dose of gamma-irradiation sufficientto reduce the bioburden of the container and the chromatography resin,where the at least one alcohol is present in an amount sufficient toameliorate the loss of binding capacity of the chromatography resinafter/upon exposure to the dose of gamma-irradiation. Also provided arereduced bioburden chromatography columns containing a reduced bioburdenchromatography resin prepared by any of the methods described herein,compositions including (i) a chromatography resin and (ii) a liquidincluding at least one alcohol, methods of performing reduced bioburdencolumn chromatography using at least one of these reduced bioburdenchromatography columns, and integrated, closed or substantially closed,and continuous processes for reduced bioburden manufacturing of apurified recombinant protein that include the use of at least one ofthese reduced bioburden chromatography columns. Any of thechromatography resins produced by any of the methods described herein,any of the packed chromatography columns produced by any of the methodsdescribed herein, any of the methods of performing columnchromatography, and any of the processes described herein can besterile, absolutely sterile, aseptic, or reduced bioburden. Any of thechromatography resins produced by any of the methods described herein,any of the chromatography columns produced by any of the methodsdescribed herein, and any of the processes described herein can beaseptic and sterile, absolutely sterile, aseptic, or reduced bioburden.

Provided herein are methods of reducing bioburden of a chromatographyresin that include: exposing a container comprising a compositioncomprising (i) a chromatography resin and (i) a liquid comprising atleast one alcohol, to a dose of gamma-irradiation sufficient to reducethe bioburden of the container and the chromatography resin, wherein theat least one alcohol is present in an amount sufficient to amelioratethe loss of binding capacity of the chromatography resin after exposureto the dose of gamma-irradiation.

In some embodiments, the method can further include, prior to exposing,disposing the composition into the container.

In some embodiments, the container is a storage vessel.

In some embodiments, the container is a chromatography column.

In some embodiments, the container is a packed chromatography column.

In some embodiments, the composition is a slurry of sedimentedchromatography resin.

In some embodiments, the composition is a wetted solid mixture.

In some embodiments of any of the methods described herein, the at leastone alcohol is selected from the group of: benzyl alcohol, cyclohexanol,isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol,propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenyl ethanol,sec-phenyl ethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol,2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol,2-phenyl-1-butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol,dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol.

In some embodiments, the at least one alcohol includes benzyl alcohol.

In some embodiments of any of the methods described herein, the totalsum concentration of the one or more alcohols in the liquid is about0.01% v/v to about 10% v/v.

In some embodiments of any of the methods described herein, the liquidcan further include at least one antioxidant agent and/or chelator.

In some embodiments, the liquid includes at least one antioxidant agentand/or chelator in an amount sufficient to ameliorate the loss ofbinding capacity of the chromatography resin after exposure to the doseof gamma-irradiation.

In some embodiments of any of the methods described herein, the liquidincludes at least one antioxidant agent selected from the groupconsisting of: reduced glutathione, reduced thioredoxin, reducedcysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol.

In some embodiments, the liquid includes at least one antioxidant agentselected from the group consisting of: mannitol, sodium ascorbate,histidine, and methionine.

In some embodiments, the liquid includes mannitol, sodium ascorbate,histidine, and methionine.

In some embodiments of any of the methods described herein, the liquidincludes: (i) between 75 mM and about 125 mM mannitol; (ii) between 75mM and about 125 mM methionine; (iii) between 75 mM and about 125 mMsodium ascorbate; (iv) between 75 mM and about 125 mM histidine; (v)between 30 mM and about 70 mM methionine and between about 30 mM andabout 70 mM histidine; (vi) between about 10 mM and about 50 mMmethionine, between about 10 mM and about 50 mM histidine, and betweenabout 10 mM and about 50 mM sodium ascorbate; or (vii) between about 5mM to about 45 mM sodium ascorbate, between about 5 mM and about 45 mMmethionine, between about 5 mM and about 45 mM mannitol, and betweenabout 5 mM to about 45 mM histidine.

In some embodiments of any of the methods described herein, the liquidis a buffered solution.

In some embodiments of any of the methods described herein, the liquidincludes at least one chelator selected from the group consisting of:ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonicacid sodium (DMPS), dimercaptosuccinic acid (DMSA), metallothionin, anddesferroxamine.

In some embodiments of any of the methods described herein, thechromatography resin is selected from the group consisting of: anionicexchange chromatography resin, cationic exchange chromatography resin,affinity chromatography resin, hydrophobic interaction chromatographyresin, and size exclusion chromatography resin.

In some embodiments, the composition includes affinity chromatographyresin including a protein ligand.

In some embodiments, the protein ligand is protein A.

In some embodiments, the composition includes an anionic exchangechromatography resin.

In some embodiments, the anionic exchange chromatography resin includesN-benzyl-N-methyl-ethanolamine groups.

In some embodiments of any of the methods described herein, the dose isbetween about 15 kGy to about 45 kGy.

In some embodiments, the dose is between about 20 kGy to about 30 kGy.

In some embodiments, the dose is between about 23 kGy and about 27 kGy.

In some embodiments of any of the methods described herein, exposing isperformed at a temperature between about −25° C. and about 0° C.,inclusive.

In some embodiments of any of the methods described herein, exposing isperformed at a temperature between about 0° C. and about 25° C.,inclusive.

Provided herein are reduced bioburden chromatography resins produced byany of the methods described herein.

In some embodiments, the resin has a sterility assurance level (SAL) ofbetween about 1×10⁻⁸ to about 1×10⁻⁵.

In some embodiments, the resin has a sterility assurance level (SAL) ofbetween about 1×10⁻⁷ to about 1×10⁻⁶.

In some embodiments of any of the resins described herein, wherein thechromatography resin includes at least one resin selected from the groupconsisting of: anionic exchange chromatography resin, cationic exchangechromatography resin, affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin.

In some embodiments, the chromatography resin includes affinitychromatography resin including a protein ligand.

In some embodiments, the protein ligand is protein A.

In some embodiments, the chromatography resin includes anionic exchangechromatography resin.

In some embodiments, the anionic exchange chromatography resin includesN-benzyl-N-methyl-ethanolamine groups.

Provided herein are methods of making a reduced bioburden packedchromatography column that include: providing any of the reducedbioburden chromatography resins described herein; and packing thechromatography resin into a reduced bioburden column in an asepticenvironment.

Provided herein are the reduced bioburden packed chromatography columnsproduced by any of the methods described herein.

Provided herein are the reduced bioburden packed chromatography columnsproduced by any of the methods described herein.

In some embodiments, the resin in the packed column has a sterilityassurance level (SAL) of between about 1×10⁻⁸ to about 1×10⁻⁵.

In some embodiments, the resin has a sterility assurance level (SAL) ofbetween about 1×10⁻⁷ to about 1×10⁻⁶.

In some embodiments of any of the resins described herein, the resin inthe packed column includes at least one resin selected from the groupconsisting of: anionic exchange chromatography resin, cationic exchangechromatography resin, affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin.

In some embodiments, the resin includes affinity or pseudo-affinitychromatography resin including a protein ligand.

In some embodiments, the ligand is protein A.

In some embodiments, the resin includes anionic exchange chromatographyresin.

In some embodiments, the anionic exchange chromatography resin includesN-benzyl-N-methyl-ethanolamine groups.

Provided herein are compositions that include (i) a chromatography resinand (ii) a liquid including at least one alcohol, wherein the at leastone alcohol is present in an amount sufficient to ameliorate the loss ofbinding capacity of the chromatography resin upon treatment with a doseof gamma-irradiation sufficient to reduce bioburden of the composition.

In some embodiments, the composition is a slurry of sedimentedchromatography resin.

In some embodiments, the composition is a wetted solid mixture.

In some embodiments of any of the compositions described herein, the atleast one alcohol is selected from the group of: benzyl alcohol,cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol,propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol,2-phenyl ethanol, sec-phenyl ethanol, 3-phenyl-1-propanol,1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol,1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol,4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and4-phenyl-1-butanol.

In some embodiments, the at least one alcohol includes benzyl alcohol.

In some embodiments of any of the compositions described herein, thetotal sum concentration of the one or more alcohols in the liquid isabout 0.01% v/v to about 10% v/v.

In some embodiments of any of the compositions described herein, theliquid further includes at least one antioxidant agent and/or chelator.

In some embodiments, the liquid further includes at least oneantioxidant agent and/or chelator in an amount sufficient to amelioratethe loss of binding capacity of the chromatography resin after exposureto the dose of gamma-irradiation.

In some embodiments of any of the compositions described herein, theliquid includes at least one antioxidant agent selected from the groupconsisting of: reduced glutathione, reduced thioredoxin, reducedcysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol.

In some embodiments, the liquid includes at least one antioxidant agentselected from the group consisting of: mannitol, sodium ascorbate,histidine, and methionine.

In some embodiments, the liquid includes mannitol, sodium ascorbate,histidine, and methionine.

In some embodiments of any of the compositions described herein, theliquid includes: (i) between 75 mM and about 125 mM mannitol; (ii)between 75 mM and about 125 mM methionine; (iii) between 75 mM and about125 mM sodium ascorbate; (iv) between 75 mM and about 125 mM histidine;(v) between 30 mM and about 70 mM methionine and between about 30 mM andabout 70 mM histidine; (vi) between about 10 mM and about 50 mMmethionine, between about 10 mM and about 50 mM histidine, and betweenabout 10 mM and about 50 mM sodium ascorbate; or (vii) between about 5mM to about 45 mM sodium ascorbate, between about 5 mM and about 45 mMmethionine, between about 5 mM and about 45 mM mannitol, and betweenabout 5 mM and about 45 mM histidine.

In some embodiments of any of the compositions described herein, theliquid is a buffered solution.

In some embodiments of any of the compositions described herein, thecomposition includes at least one chelator selected from the groupconsisting of: ethylenediaminetetraacetic acid (EDTA),2,3-dimercapto-1-propanesulfonic acid sodium (DMPS), dimercaptosuccinicacid (DMSA), metallothionin, and desferroxamine.

In some embodiments of any of the compositions described herein, thechromatography resin includes at least one resin selected from the groupconsisting of: anionic exchange chromatography resin, cationic exchangechromatography resin, affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin.

In some embodiments of any of the compositions described herein, theresin includes affinity chromatography resin including a protein ligand.

In some embodiments, the protein ligand is protein A.

Provided herein are methods of performing reduced bioburden columnchromatography that include: (a) providing any of the reduced bioburdenpacked chromatography columns described herein; and (b) performingcolumn chromatography using the reduced bioburden packed chromatographycolumn and reduced bioburden buffer in a closed system.

In some embodiments, reduced bioburden column chromatography using thereduced bioburden packed chromatography column is performed continuouslyfor a period of at least 4 days.

In some embodiments, reduced bioburden column chromatography using thereduced bioburden packed chromatography column is performed continuouslyfor a period of at least 5 days.

In some embodiments, the reduced bioburden column chromatography usingthe reduced bioburden packed chromatography column is performedcontinuously for a period of at least 7 days.

In some embodiments, the reduced bioburden column chromatography usingthe reduced bioburden packed chromatography column is performedcontinuously for a period of at least 14 days.

In some embodiments, the reduced bioburden column chromatography usingthe reduced bioburden packed chromatography column is performedcontinuously for a period of at least 28 days.

In some embodiments, the resin in the reduced bioburden packedchromatography column in (a) has a percentage binding capacity ofbetween about 75% and about 100% as compared to the same resin nottreated with gamma-irradiation.

In some embodiments, the resin in the reduced bioburden packedchromatography column includes at least one resin selected from thegroup consisting of: anionic exchange chromatography resin, cationicexchange chromatography resin, affinity chromatography resin,hydrophobic interaction chromatography resin, and size exclusionchromatography resin.

In some embodiments, the resin includes affinity chromatography resinincluding a protein ligand.

In some embodiments, the protein ligand is protein A.

In some embodiments, the resin includes anionic exchange chromatographyresin.

Provided herein are integrated, closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant protein thatinclude: (a) providing a liquid culture medium comprising a recombinantprotein that is substantially free of cells; and (b) continuouslyfeeding the liquid culture medium into a multi-column chromatographysystem (MCCS) comprising at least one of any of the reduced bioburdenpacked chromatography columns described herein; wherein the processutilizes reduced bioburden buffer, is integrated, and runs continuouslyfrom the liquid culture medium to an eluate from the MCCS that is thepurified recombinant protein.

In some embodiments, the MCCS performs at least two different unitoperations.

In some embodiments, the process includes column switching.

In some embodiments, the MCCS performs the unit operations of capturingthe recombinant protein and inactivating viruses.

In some embodiments, the MCCS performs the unit operations of capturingand purifying the recombinant protein.

In some embodiments, the MCCS includes at least two reduced bioburdenpacked chromatography columns.

In some embodiments, the MCCS is a periodic counter currentchromatography system.

In some embodiments, the MCCS includes a plurality of columns foraffinity or pseudo-affinity chromatography, cation exchangechromatography, anion exchange chromatography, or size exclusionchromatography, or any combination thereof.

In some embodiments, the MCCS includes a column for affinitychromatography, and affinity chromatography is performed in the processwith a capture mechanism selected from the group consisting of: proteinA-binding capture mechanism, substrate-binding capture mechanism,antibody- or antibody fragment-binding capture mechanism,aptamer-binding capture mechanism, and cofactor-binding capturemechanism.

In some embodiments, the affinity chromatography is performed in theprocess with a protein A-binding capture mechanism, and the recombinantprotein is an antibody or an antibody fragment.

Provided herein are integrated, closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant protein thatinclude: (a) providing a liquid culture medium comprising a recombinantprotein that is substantially free of cells; (b) continuously feedingthe liquid culture medium into a first multi-column chromatographysystem (MCCS1); (c) capturing the recombinant protein in the liquidculture medium using the MCCS1; (d) producing an eluate from the MCCS1that includes the recombinant protein and continuously feeding theeluate into a second multi-column chromatography system (MCCS2); (e)continuously feeding the recombinant protein from the eluate into theMCCS2 and subsequently eluting the recombinant protein to therebyproduce the purified recombinant protein, wherein: the process utilizesreduced bioburden buffer, is integrated, and runs continuously from theliquid culture medium to the purified recombinant protein, and at leastone column in the MCCS1 and/or MCCS2 contains any of the reducedbioburden packed chromatography columns described herein.

In some embodiments, the MCCS1 and/or the MCCS2 performs at least twodifferent unit operations.

In some embodiments, the process involves column switching.

In some embodiments, the MCCS1 performs the unit operations of capturingthe recombinant therapeutic protein and inactivating viruses.

In some embodiments, the MCCS2 performs the unit operations of purifyingand polishing the recombinant protein.

In some embodiments, the MCCS1 and/or MCCS2 include at least twochromatography columns.

In some embodiments, the MCCS1 is a first periodic counter currentchromatography system (PCCS1).

In some embodiments, the capturing is performed using affinitychromatography, cation exchange chromatography, anion exchangechromatography, or size exclusion chromatography, or any combinationthereof.

In some embodiments, the affinity chromatography is performed with acapture mechanism selected from the group consisting of: proteinA-binding capture mechanism, substrate-binding capture mechanism,antibody- or antibody fragment-binding capture mechanism,aptamer-binding capture mechanism, and cofactor-binding capturemechanism.

In some embodiments, the affinity chromatography is performed with aprotein-A binding capture mechanism, and the recombinant protein is anantibody or an antibody fragment.

In some embodiments, the MCCS2 is a second periodic counter current(PCCS2) chromatography system.

In some embodiments of any of the processes described herein, therecombinant protein is a therapeutic recombinant protein.

In some embodiments of any of the processes described herein, theprocess further includes formulating the purified therapeuticrecombinant protein into a pharmaceutical composition.

In some embodiments of any of the processes described herein, theprocess is performed continuously for a period of at least 4 days.

In some embodiments, the process is performed continuously for a periodof at least 5 days.

In some embodiments, the process is performed continuously for a periodof at least 7 days.

In some embodiments, the process is performed continuously for a periodof at least 14 days.

In some embodiments, the process is performed continuously for a periodof at least 28 days.

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a reduced bioburdenchromatography column” represents “one or more reduced bioburdenchromatography columns.”

The term “bioburden” is art known and refers to the level ofself-replicating biological contaminants present in a composition (e.g.,solid or liquid) and/or on the surface (e.g., exterior and/or interiorsurface) of an article(s). For example, bioburden can refer toself-replicating biological contaminants present in a compositioncontaining a chromatography resin or a packed chromatography resin(e.g., self-replicating biological contaminants present in a packedchromatography resin in a packed chromatography column). In otherexamples, bioburden can to refer to self-replicating biologicalcontaminants on the inner surface of a chromatography column and/orwithin the chromatography resin within the chromatography column (e.g.,biological contaminants on the inner surface of a chromatography columnand biological contaminants in the packed chromatography resin withinthe chromatography column). Bioburden can also refer to theself-replicating biological contaminants present within a liquid (e.g.,a buffer used in any of the methods or processes described herein).Non-limiting examples of self-replicating biological contaminants can bebacteria (e.g., Gram-positive or Gram-negative bacteria, or a bacterialspore), mycobacteria, viruses (e.g., a vesivirus, a Cache Valley virus,a parvovirus, a herpes virus, and a bunyavirus), parasites, fungi,yeast, and protozoa. Exemplary methods for determining bioburden aredescribed herein. Additional methods for determining bioburden are knownin the art.

The term “reducing bioburden” is art known and refers to a decrease(e.g., a detectable decrease) in the level of self-replicatingbiological contaminants present in a composition (e.g., solid or liquid)and/or on the surface (e.g., exterior and/or interior surface) of anarticle(s). Non-limiting examples of methods for reducing bioburden of achromatography resin (e.g., packed chromatography resin), buffer, and/ora chromatography column (e.g., a packed chromatography column) aredescribed herein. Additional methods for reducing bioburden of any ofthe compositions described herein are known in the art.

The term “reduced bioburden chromatography resin” means a chromatographyresin that has been treated to decrease the level of self-replicatingbiological contaminants present in the chromatography resin (e.g., adetectable decrease in the level of self-replicating biologicalcontaminants present in a composition containing a chromatography resin,e.g., a slurry). For example, reduced bioburden chromatography resin canbe a resin exposed to gamma-irradiation in a dose sufficient to decreasethe level of self-replicating biological contaminants in thechromatography resin (e.g., a composition containing a chromatographyresin exposed to gamma-irradiation in a dose sufficient to decrease thelevel of self-replicating biological contaminants in the chromatographyresin). For example, a reduced bioburden chromatography resin can be aresin that has been exposed to a dose of between about 1 kGy to about 15kGy, between about 1 kGy and about 20 kGy gamma-irradiation, betweenabout 1 kGy and about 25 kGy gamma-irradiation, between about 1 kGy andabout 30 kGy gamma-irradiation, or between about 1 kGy and about 35 kGygamma-irradiation. Exemplary methods for reducing bioburden of achromatography resin are described herein. Additional methods forreducing the bioburden of a chromatography resin are known in the art.

The term “reduced bioburden chromatography column” means achromatography column (e.g., a packed chromatography column) thatincludes a treated chromatography resin (e.g., gamma-irradiatedchromatography resin), that has a level of self-replicating biologicalcontaminants that is less than the level of self-replicating biologicalcontaminants present in an identical chromatography column that includesan untreated chromatography resin. For example, a reduced bioburdenchromatography column can include a treated chromatography resin havinga sterility assurance level of at least or about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸,1×10⁻⁹, or 1×10⁻¹⁰.

The term “reduced bioburden buffer” is art known and means a treated(e.g., filtered, autoclaved, and/or gamma-irradiated) liquid (e.g., atreated buffered solution) that has a level of self-replicatingcontaminating agent(s) that is less than the level of self-replicatingcontaminating agent(s) found in an identical untreated liquid. Forexample, a reduced bioburden buffer can have a sterility assurance levelof at least or about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹, or 1×10⁻¹⁰.

“Absolute sterility” or “absolutely sterile” are terms used to describea composition or process that is/are completely free of self-replicatingbiological contaminants. For example, the term can apply to agamma-irradiated chromatography resin, the interior surface and contents(e.g., chromatography resin) of a chromatography column, and/or abuffer. An absolutely sterile composition or process can be clean (asthat term is known in the art).

“Sterile” or “sterility” are terms used to describe a composition orprocess that have a sterility assurance level of about or less than1.0×10⁻⁶ (e.g., about or less than 1.0×10⁻⁷, about or less than1.0×10⁻⁸, about or less than 1.0×10⁻⁹, or 1×10⁻¹⁰). The determination ofwhether a composition or process is sterile can be tested using a numberof validated production processes known in the art. For example, asterile composition or process can be completely free of viableself-replicating biological contaminants (e.g., any of theself-replicating biological contaminants described herein). A sterilecomposition or process can also be clean (as that term is known in theart).

The term “sterilization” means a validated process used to render acomposition sterile (as defined herein). The inactivation rate ofresistant indicator self-replicating biological contaminants (e.g.,bacteria) during a treatment process can be measured in order todetermine whether sterility (as defined herein) has been achieved for acomposition.

The term “sterility assurance level” or “SAL” is art-known and means alevel of confidence of achieving absolute sterility within a batch oftreated units. The probability is usually calculated based on theresults of inactivation studies performed during validation andexpressed in the form of 1×10^(−n).

The term “aseptic” is used to describe a composition or process that isfree of disease-causing or symptom-causing self-replicating biologicalcontaminants (e.g., any of the self-replicating biological contaminantsdescribed herein). An aseptic composition or process can also be clean(as that term is known in the art).

The term “unit operation” is a term of art and means a functional stepthat can be performed in a process of purifying a recombinant proteinfrom a liquid culture medium. For example, a unit of operation can befiltering (e.g., removal of contaminant bacteria, yeast, viruses, and/ormycobacteria, and/or particulate matter from a fluid including arecombinant protein), capturing, epitope tag removal, purifying, holdingor storing, polishing, virus inactivating, adjusting the ionicconcentration and/or pH of a fluid including the recombinant protein,and removing unwanted salts.

The term “capturing” means a step performed to partially purify orisolate (e.g., at least or about 5%, e.g., at least or about 10%, 15%,20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orat least or about 95% pure by weight) and concentrate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more othercomponents present in a liquid culture medium or a diluted liquidculture medium (e.g., culture medium proteins or one or more othercomponents (e.g., DNA, RNA, or other proteins) present in or secretedfrom a mammalian cell). Typically, capturing is performed using achromatography resin that binds a recombinant protein (e.g., through theuse of affinity chromatography). Non-limiting methods for capturing arecombinant protein from a liquid culture medium or diluted liquidculture medium are described herein and others are known in the art. Arecombinant protein can be captured from a liquid culture medium usingat least one chromatography column and/or chromatographic membrane(e.g., any of the chromatography columns and/or chromatographicmembranes described herein).

The term “purifying” means a step performed to isolate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more otherimpurities (e.g., bulk impurities) or components present in a fluidincluding a recombinant protein (e.g., liquid culture medium proteins orone or more other components (e.g., DNA, RNA, other proteins,endotoxins, viruses, etc.) present in or secreted from a mammaliancell). For example, purifying can be performed during or after aninitial capturing step. Purification can be performed using achromatography resin, membrane, or any other solid support that bindseither a recombinant protein or contaminants (e.g., through the use ofaffinity chromatography, hydrophobic interaction chromatography, anionor cation exchange chromatography, or molecular sieve chromatography). Arecombinant protein can be purified from a fluid including therecombinant protein using at least one chromatography column and/orchromatographic membrane (e.g., any of the chromatography columns orchromatographic membranes described herein).

The term “polishing” is a term of art and means a step performed toremove remaining trace or small amounts of contaminants or impuritiesfrom a fluid including a recombinant protein (e.g., a recombinanttherapeutic protein) that is close to a final desired purity. Forexample, polishing can be performed by passing a fluid including therecombinant protein through a chromatographic column(s) or membraneabsorber(s) that selectively binds to either the target recombinantprotein or small amounts of contaminants or impurities present in afluid including a recombinant protein. In such an example, theeluate/filtrate of the chromatographic column(s) or membrane absorber(s)includes the recombinant protein.

The term “filtering” means the removal of at least part of (e.g., atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99%) undesired biologicalcontaminants (e.g., a mammalian cell, bacteria, yeast cells, viruses, ormycobacteria) and/or particulate matter (e.g., precipitated proteins)from a liquid (e.g., a liquid culture medium or fluid present in any ofthe processes described herein).

The term “eluate/filtrate” is a term of art and means a fluid that isemitted from a chromatography column or chromatographic membrane thatincludes a detectable amount of a recombinant protein (e.g., arecombinant therapeutic protein).

The term “integrated process” means a process which is performed usingstructural elements that function cooperatively to achieve a specificresult (e.g., the purification of a recombinant protein from a liquidculture medium).

The term “continuous process” means a process which continuously feedsfluid through at least a part of the system. For example, a continuousprocess is a process which continuously feeds a liquid culture mediumincluding a recombinant protein from a bioreactor through a MCCS.Another example of a continuous process is a process which continuouslyfeeds a liquid culture medium including a recombinant protein from abioreactor through a first and second MCCS (MCCS1 and MCCS2). Additionalexamples include a process which continuously feeds a liquid culturemedium including a recombinant protein through a MCCS, a process thatcontinuously feeds a liquid culture medium including a recombinantprotein through a MCCS1 and a MCCS2, or a process that continuouslyfeeds a fluid including a recombinant protein through a MCCS2.

The term “closed process” is a term of art and means a process that isperformed such that components of the process (e.g., chromatographyresins and/or buffers) that come into contact with the recombinantprotein or liquids including the recombinant protein are notintentionally exposed to contaminating agents for a significant periodof time (e.g., not intentionally air-exposed for a significant period oftime).

The term “therapeutic protein drug substance” means a recombinantprotein (e.g., an immunoglobulin, protein fragment, engineered protein,or enzyme) that has been sufficiently purified or isolated fromcontaminating proteins, lipids, and nucleic acids (e.g., contaminatingproteins, lipids, and nucleic acids present in a liquid culture mediumor from a host cell (e.g., from a mammalian, yeast, or bacterial hostcell)) and biological contaminants (e.g., viral and bacterialcontaminants), and can be formulated into a pharmaceutical agent withoutany further substantial purification and/or decontamination step(s).

The term “multi-column chromatography system” or “MCCS” means a systemof a total of two or more interconnected or switching chromatographycolumns and/or chromatographic membranes. A non-limiting example of amulti-column chromatography system is a periodic counter currentchromatography system (PCC) including a total of two or moreinterconnected or switching chromatography columns and/orchromatographic membranes. Additional examples of multi-columnchromatography systems are described herein and are known in the art.

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specified substance (e.g., a mammalian cell or acontaminating protein, nucleic acid, carbohydrate, or lipid form amammalian cell).

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). For example, a mammalian cell can be an immortalized cell.In some embodiments, the mammalian cell is a differentiated cell. Insome embodiments, the mammalian cell is an undifferentiated cell.Non-limiting examples of mammalian cells are described herein.Additional examples of mammalian cells are known in the art.

The term “culturing” or “cell culturing” means the maintenance orproliferation of a mammalian cell under a controlled set of physicalconditions.

The term “culture of mammalian cells” means a liquid culture mediumincluding a plurality of mammalian cells that is maintained orproliferated under a controlled set of physical conditions.

The term “liquid culture medium” means a fluid that includes sufficientnutrients to allow a cell (e.g., a mammalian cell) to grow orproliferate in vitro. For example, a liquid culture medium can includeone or more of: amino acids (e.g., 20 amino acids), a purine (e.g.,hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol,thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine,riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium,glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.In some embodiments, a liquid culture medium can include serum from amammal. In some embodiments, a liquid culture medium does not includeserum or another extract from a mammal (a defined liquid culturemedium). In some embodiments, a liquid culture medium can include tracemetals, a mammalian growth hormone, and/or a mammalian growth factor.Another example of liquid culture medium is minimal medium (e.g., amedium including only inorganic salts, a carbon source, and water).Non-limiting examples of liquid culture medium are described herein.Additional examples of liquid culture medium are known in the art andare commercially available. A liquid culture medium can include anydensity of mammalian cells. For example, as used herein, a volume ofliquid culture medium removed from a bioreactor can be substantiallyfree of mammalian cells.

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not include any components (e.g.,proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not include a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that includes a mammalian serum.

The term “chemically-defined liquid culture medium” is a term of art andmeans a liquid culture medium in which all of the chemical componentsare known. For example, a chemically-defined liquid culture medium doesnot include fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically include a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not include any protein (e.g., any detectable protein).

The term “immunoglobulin” means a polypeptide including an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, and/or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin, e.g., at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM), e.g., asubclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The immunoglobulinmay be an antibody fragment, e.g., a Fab fragment, a F(ab′)₂ fragment,or a scFv fragment. The immunoglobulin may also be a bi-specificantibody or a tri-specific antibody, or a dimer, trimer, or multimerantibody, or a diabody, an Affibody®, or a Nanobody®. The immunoglobulincan also be an engineered protein including at least one immunoglobulindomain (e.g., a fusion protein). Non-limiting examples ofimmunoglobulins are described herein and additional examples ofimmunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, at leastor about 5 amino acids, at least or about 6 amino acids, at least orabout 7 amino acids, at least or about 8 amino acids, at least or about9 amino acids, at least or about 10 amino acids, at least or about 11amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the processes described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that include at least one recombinantscaffolding sequence.

The term “secreted protein” or “secreted recombinant protein” means aprotein (e.g., a recombinant protein) that originally included at leastone secretion signal sequence when it is translated within a mammaliancell, and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is secreted at least partiallyinto the extracellular space (e.g., a liquid culture medium). Skilledpractitioners will appreciate that a “secreted” protein need notdissociate entirely from the cell to be considered a secreted protein.

The term “perfusion bioreactor” means a bioreactor including a pluralityof cells (e.g., mammalian cells) in a first liquid culture medium,wherein the culturing of the cells present in the bioreactor includesperiodic or continuous removal of the first liquid culture medium and atthe same time or shortly thereafter adding substantially the same volumeof a second liquid culture medium to the bioreactor. In some examples,there is an incremental change (e.g., increase or decrease) in thevolume of the first liquid culture medium removed and added overincremental periods (e.g., an about 24-hour period, a period of betweenabout 1 minute and about 24-hours, or a period of greater than 24 hours)during the culturing period (e.g., the culture medium refeed rate on adaily basis). The fraction of media removed and replaced each day canvary depending on the particular cells being cultured, the initialseeding density, and the cell density at a particular time. “RV” or“reactor volume” means the volume of the culture medium present at thebeginning of the culturing process (e.g., the total volume of theculture medium present after seeding).

The term “fed-batch bioreactor” is a term of art and means a bioreactorincluding a plurality of cells (e.g., mammalian cells) in a first liquidculture medium, wherein the culturing of the cells present in thebioreactor includes the periodic or continuous addition of a secondliquid culture medium to the first liquid culture medium withoutsubstantial or significant removal of the first liquid culture medium orsecond liquid culture medium from the cell culture. The second liquidculture medium can be the same as the first liquid culture medium. Insome examples of fed-batch culture, the second liquid culture medium isa concentrated form of the first liquid culture medium. In some examplesof fed-batch culture, the second liquid culture medium is added as a drypowder.

The term “clarified liquid culture medium” means a liquid culture mediumobtained from a bacterial or yeast cell culture that is substantiallyfree (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) ofbacteria or yeast cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the percentage binding capacity (as comparedto non-irradiated chromatography resin loaded with the same material) ofMabSelect™ SuRe™ (Protein A chromatography resin) in multiple cycles ofchromatography following 40-49 kGy irradiation in the presence of one ofthe following buffers: (i) 25 mM sodium ascorbate, 25 mM methionine, 25mM histidine, and 25 mM mannitol in 50 mM sodium phosphate buffer(“SMM'H”); (ii) 2% v/v benzyl alcohol (“2% BA”); and (iii) 25 mM sodiumascorbate, 25 mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v/vbenzyl alcohol in 50 mM sodium phosphate buffer (“SMM'H+2% BA”).

FIG. 2 is a graph showing the percentage binding capacity (as comparedto non-irradiated chromatography resin loaded with the same material) ofCapto Adhere chromatography resin in multiple cycles of chromatographyfollowing 28-34 kGy or 40-49 kGy irradiation (as described in Example 2)in the presence of one of the following buffers: (i) 25 mM sodiumascorbate, 25 mM methionine, 25 mM histidine, and 25 mM mannitol in 50mM sodium phosphate buffer (“SMM'H”); or (ii) 25 mM sodium ascorbate, 25mM methionine, 25 mM histidine, 25 mM mannitol, and 2% v/v benzylalcohol in 50 mM sodium phosphate buffer (“SMM'H+2% BA”).

DETAILED DESCRIPTION

Provided herein are methods of reducing bioburden of a chromatographyresin that include exposing a container including a compositionincluding (i) a chromatography resin and (ii) a liquid including atleast one (e.g., two, three, four, or five) alcohol to a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin, where the at least one alcohol is presentin an amount sufficient to ameliorate the loss of binding capacity ofthe chromatography resin after/upon exposure to the dose ofgamma-irradiation. Also provided are reduced bioburden chromatographycolumns containing a reduced bioburden chromatography resin prepared byany of the methods described herein, compositions including (i) achromatography resin and (ii) a liquid including at least one (e.g.,two, three, four, or five) alcohol, methods of performing reducedbioburden column chromatography using at least one of these reducedbioburden chromatography columns, and integrated, closed orsubstantially closed, and continuous processes for reduced bioburdenmanufacturing of a purified recombinant protein that include the use ofat least one of these reduced bioburden chromatography columns.Non-limiting aspects of these methods and processes are described below.As can be appreciated in the art, the various aspects described belowcan be used in any combination without limitation.

Compositions Containing Chromatography Resin and at Least One Alcohol

Provided herein are compositions including (i) a chromatography resin(e.g., any of the chromatography resins described herein or known in theart) and (ii) a liquid including at least one (e.g., two, three, four,or five) alcohol (e.g., any of the exemplary alcohols described hereinor known in the art), wherein the at least one alcohol is present in anamount sufficient to ameliorate the loss of binding capacity of thechromatography resin upon treatment with a dose of gamma-irradiationsufficient to reduce bioburden of the composition. For example, thechromatography resin can be at least one of anionic exchangechromatography resin, cationic exchange chromatography resin, affinityor pseudo-affinity chromatography resin, hydrophobic interactionchromatography resin, and size exclusion chromatography resin, or anycombination thereof In some examples, the chromatography resin is aresin including a protein or peptide ligand (e.g., an affinitychromatography resin with a protein or peptide ligand, e.g., a protein Aor protein G chromatography resin).

The composition, e.g., can be a slurry of sedimented chromatographyresin. In some examples, the composition can be a wetted or moist solidmixture. In some examples, the composition is a chromatography resinpacked in the liquid.

In some examples of any of the compositions, the at least one (e.g.,two, three, four, or five) alcohol can be selected from the group of:benzyl alcohol, cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol,methanol, ethanol, propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol,hexadecan-1-ol, 2-phenyl ethanol, sec-phenyl ethanol,3-phenyl-1-propanol, 1-phenyl-1-propanol, 2-phenyl-1-propanol,2-phenyl-2-propanol, 1-phenyl-2-butanol, 2-phenyl-1-butanol,3-phenyl-1-butanol, 4-phenyl-2-butanol, dl-1-phenyl-2-pentanol,5-phenyl-1-pentanol, and 4-phenyl-1-butanol. In some examples, the atleast one alcohol can be benzyl alcohol.

In some examples of any of the compositions, the total sum concentrationof the one or more alcohols in the liquid or the composition is about0.01% v/v to about 20% v/v, about 0.01% v/v to about 19% v/v, about0.01% v/v to about 18% v/v, about 0.01% v/v to about 17% v/v, about0.01% v/v to about 16% v/v, about 0.01% v/v to about 15% v/v, about0.01% v/v to about 14% v/v, about 0.01% v/v to about 13% v/v, about0.01% v/v to about 12% v/v, about 0.01% v/v to about 11% v/v, about0.01% v/v to about 10% v/v, about 0.01% v/v to about 9% v/v, about 0.01%v/v to about 8% v/v, about 0.01% v/v to about 7% v/v, about 0.01% v/v toabout 6% v/v, about 0.01% v/v to about 5% v/v, about 0.01% v/v to about4.5% v/v, about 0.01% v/v to about 4.0% v/v, about 0.01% v/v to about3.5% v/v, about 0.01% v/v to about 3.0% v/v, about 0.01% v/v to about2.5% v/v, about 0.01% v/v to about 2.2% v/v, about 0.01% v/v to about2.0% v/v, about 0.01% v/v to about 1.8% v/v, about 0.01% v/v to about1.6% v/v, about 0.01% v/v to about 1.4% v/v, about 0.01% v/v to about1.2% v/v, about 0.01% v/v to about 1.0% v/v, about 0.01% v/v to about0.8% v/v, about 0.01% v/v to about 0.6% v/v, about 0.01% v/v to about0.4% v/v, about 0.01% v/v to about 0.2% v/v, about 0.01% v/v to about0.1% v/v, about 0.01% v/v to about 0.05% v/v, about 0.05% v/v to about20% v/v, about 0.05% v/v to about 19% v/v, about 0.05% v/v to about 18%v/v, about 0.05% v/v to about 17% v/v, about 0.05% v/v to about 16% v/v,about 0.05% v/v to about 15% v/v, about 0.05% v/v to about 14% v/v,about 0.05% v/v to about 13% v/v, about 0.05% v/v to about 12% v/v,about 0.05% v/v to about 11% v/v, about 0.05% v/v to about 10% v/v,about 0.05% v/v to about 9% v/v, about 0.05% v/v to about 8% v/v, about0.05% v/v to about 7% v/v, about 0.05% v/v to about 6% v/v, about 0.05%v/v to about 5% v/v, about 0.05% v/v to about 4.5% v/v, about 0.05% v/vto about 4.0% v/v, about 0.05% v/v to about 3.5% v/v, about 0.05% v/v toabout 3.0% v/v, about 0.05% v/v to about 2.5% v/v, about 0.05% v/v toabout 2.2% v/v, about 0.05% v/v to about 2.0% v/v, about 0.05% v/v toabout 1.8% v/v, about 0.05% v/v to about 1.6% v/v, about 0.05% v/v toabout 1.4% v/v, about 0.05% v/v to about 1.2% v/v, about 0.05% v/v toabout 1.0% v/v, about 0.05% v/v to about 0.8% v/v, about 0.05% v/v toabout 0.6% v/v, about 0.05% v/v to about 0.4% v/v, about 0.05% v/v toabout 0.2% v/v, about 0.05% v/v to about 0.1% v/v, about 0.1% v/v toabout 20% v/v, about 0.1% v/v to about 19% v/v, about 0.1% v/v to about18% v/v, about 0.1% v/v to about 17% v/v, about 0.1% v/v to about 16%v/v, about 0.1% v/v to about 15% v/v, about 0.1% v/v to about 14% v/v,about 0.1% v/v to about 13% v/v, about 0.1% v/v to about 12% v/v, about0.1% v/v to about 11% v/v, about 0.1% v/v to about 10% v/v, about 0.1%v/v to about 9% v/v, about 0.1% v/v to about 8% v/v, about 0.1% v/v toabout 7% v/v, about 0.1% v/v to about 6% v/v, about 0.1% v/v to about 5%v/v, about 0.1% v/v to about 4.5% v/v, about 0.1% v/v to about 4.0% v/v,about 0.1% v/v to about 3.5% v/v, about 0.1% v/v to about 3.0% v/v,about 0.1% v/v to about 2.5% v/v, about 0.1% v/v to about 2.2% v/v,about 0.1% v/v to about 2.0% v/v, about 0.1% v/v to about 1.8% v/v,about 0.1% v/v to about 1.6% v/v, about 0.1% v/v to about 1.4% v/v,about 0.1% v/v to about 1.2% v/v, about 0.1% v/v to about 1.0% v/v,about 0.1% v/v to about 0.8% v/v, about 0.1% v/v to about 0.6% v/v,about 0.1% v/v to about 0.4% v/v, about 0.1% v/v to about 0.2% v/v,about 0.2% v/v to about 20% v/v, about 0.2% v/v to about 19% v/v, about0.2% v/v to about 18% v/v, about 0.2% v/v to about 17% v/v, about 0.2%v/v to about 16% v/v, about 0.2% v/v to about 15% v/v, about 0.2% v/v toabout 14% v/v, about 0.2% v/v to about 13% v/v, about 0.2% v/v to about12% v/v, about 0.2% v/v to about 11% v/v, about 0.2% v/v to about 10%v/v, about 0.2% v/v to about 9% v/v, about 0.2% v/v to about 8% v/v,about 0.2% v/v to about 7% v/v, about 0.2% v/v to about 6% v/v, about0.2% v/v to about 5% v/v, about 0.2% v/v to about 4.5% v/v, about 0.2%v/v to about 4.0% v/v, about 0.2% v/v to about 3.5% v/v, about 0.2% v/vto about 3.0% v/v, about 0.2% v/v to about 2.5% v/v, about 0.2% v/v toabout 2.2% v/v, about 0.2% v/v to about 2.0% v/v, about 0.2% v/v toabout 1.8% v/v, about 0.2% v/v to about 1.6% v/v, about 0.2% v/v toabout 1.4% v/v, about 0.2% v/v to about 1.2% v/v, about 0.2% v/v toabout 1.0% v/v, about 0.2% v/v to about 0.8% v/v, about 0.2% v/v toabout 0.6% v/v, about 0.2% v/v to about 0.4% v/v, about 0.4% v/v toabout 20% v/v, about 0.4% v/v to about 19% v/v, about 0.4% v/v to about18% v/v, about 0.4% v/v to about 17% v/v, about 0.4% v/v to about 16%v/v, about 0.4% v/v to about 15% v/v, about 0.4% v/v to about 14% v/v,about 0.4% v/v to about 13% v/v, about 0.4% v/v to about 12% v/v, about0.4% v/v to about 11% v/v, about 0.4% v/v to about 10% v/v, about 0.4%v/v to about 9% v/v, about 0.4% v/v to about 8% v/v, about 0.4% v/v toabout 7% v/v, about 0.4% v/v to about 6% v/v, about 0.4% v/v to about 5%v/v, about 0.4% v/v to about 4.5% v/v, about 0.4% v/v to about 4.0% v/v,about 0.4% v/v to about 3.5% v/v, about 0.4% v/v to about 3.0% v/v,about 0.4% v/v to about 2.5% v/v, about 0.4% v/v to about 2.2% v/v,about 0.4% v/v to about 2.0% v/v, about 0.4% v/v to about 1.8% v/v,about 0.4% v/v to about 1.6% v/v, about 0.4% v/v to about 1.4% v/v,about 0.4% v/v to about 1.2% v/v, about 0.4% v/v to about 1.0% v/v,about 0.4% v/v to about 0.8% v/v, about 0.4% v/v to about 0.6% v/v,about 0.6% v/v to about 20% v/v, about 0.6% v/v to about 19% v/v, about0.6% v/v to about 18% v/v, about 0.6% v/v to about 17% v/v, about 0.6%v/v to about 16% v/v, about 0.6% v/v to about 15% v/v, about 0.6% v/v toabout 14% v/v, about 0.6% v/v to about 13% v/v, about 0.6% v/v to about12% v/v, about 0.6% v/v to about 11% v/v, about 0.6% v/v to about 10%v/v, about 0.6% v/v to about 9% v/v, about 0.6% v/v to about 8% v/v,about 0.6% v/v to about 7% v/v, about 0.6% v/v to about 6% v/v, about0.6% v/v to about 5% v/v, about 0.6% v/v to about 4.5% v/v, about 0.6%v/v to about 4.0% v/v, about 0.6% v/v to about 3.5% v/v, about 0.6% v/vto about 3.0% v/v, about 0.6% v/v to about 2.5% v/v, about 0.6% v/v toabout 2.2% v/v, about 0.6% v/v to about 2.0% v/v, about 0.6% v/v toabout 1.8% v/v, about 0.6% v/v to about 1.6% v/v, about 0.6% v/v toabout 1.4% v/v, about 0.6% v/v to about 1.2% v/v, about 0.6% v/v toabout 1.0% v/v, about 0.6% v/v to about 0.8% v/v, about 0.8% v/v toabout 20% v/v, about 0.8% v/v to about 19% v/v, about 0.8% v/v to about18% v/v, about 0.8% v/v to about 17% v/v, about 0.8% v/v to about 16%v/v, about 0.8% v/v to about 15% v/v, about 0.8% v/v to about 14% v/v,about 0.8% v/v to about 13% v/v, about 0.8% v/v to about 12% v/v, about0.8% v/v to about 11% v/v, about 0.8% v/v to about 10% v/v, about 0.8%v/v to about 9% v/v, about 0.8% v/v to about 8% v/v, about 0.8% v/v toabout 7% v/v, about 0.8% v/v to about 6% v/v, about 0.8% v/v to about 5%v/v, about 0.8% v/v to about 4.5% v/v, about 0.8% v/v to about 4.0% v/v,about 0.8% v/v to about 3.5% v/v, about 0.8% v/v to about 3.0% v/v,about 0.8% v/v to about 2.5% v/v, about 0.8% v/v to about 2.2% v/v,about 0.8% v/v to about 2.0% v/v, about 0.8% v/v to about 1.8% v/v,about 0.8% v/v to about 1.6% v/v, about 0.8% v/v to about 1.4% v/v,about 0.8% v/v to about 1.2% v/v, about 0.8% v/v to about 1.0% v/v,about 1.0% v/v to about 20% v/v, about 1.0% v/v to about 19% v/v, about1.0% v/v to about 18% v/v, about 1.0% v/v to about 17% v/v, about 1.0%v/v to about 16% v/v, about 1.0% v/v to about 15% v/v, about 1.0% v/v toabout 14% v/v, about 1.0% v/v to about 13% v/v, about 1.0% v/v to about12% v/v, about 1.0% v/v to about 11% v/v, about 1.0% v/v to about 10%v/v, about 1.0% v/v to about 9% v/v, about 1.0% v/v to about 8% v/v,about 1.0% v/v to about 7% v/v, about 1.0% v/v to about 6% v/v, about1.0% v/v to about 5% v/v, about 1.0% v/v to about 4.5% v/v, about 1.0%v/v to about 4.0% v/v, about 1.0% v/v to about 3.5% v/v, about 1.0% v/vto about 3.0% v/v, about 1.0% v/v to about 2.5% v/v, about 1.0% v/v toabout 2.2% v/v, about 1.0% v/v to about 2.0% v/v, about 1.0% v/v toabout 1.8% v/v, about 1.0% v/v to about 1.6% v/v, about 1.0% v/v toabout 1.4% v/v, about 1.0% v/v to about 1.2% v/v, about 1.2% v/v toabout 20% v/v, about 1.2% v/v to about 19% v/v, about 1.2% v/v to about18% v/v, about 1.2% v/v to about 17% v/v, about 1.2% v/v to about 16%v/v, about 1.2% v/v to about 15% v/v, about 1.2% v/v to about 14% v/v,about 1.2% v/v to about 13% v/v, about 1.2% v/v to about 12% v/v, about1.2% v/v to about 11% v/v, about 1.2% v/v to about 10% v/v, about 1.2%v/v to about 9% v/v, about 1.2% v/v to about 8% v/v, about 1.2% v/v toabout 7% v/v, about 1.2% v/v to about 6% v/v, about 1.2% v/v to about 5%v/v, about 1.2% v/v to about 4.5% v/v, about 1.2% v/v to about 4.0% v/v,about 1.2% v/v to about 3.5% v/v, about 1.2% v/v to about 3.0% v/v,about 1.2% v/v to about 2.5% v/v, about 1.2% v/v to about 2.2% v/v,about 1.2% v/v to about 2.0% v/v, about 1.2% v/v to about 1.8% v/v,about 1.2% v/v to about 1.6% v/v, about 1.2% v/v to about 1.4% v/v,about 1.4% v/v to about 20% v/v, about 1.4% v/v to about 19% v/v, about1.4% v/v to about 18% v/v, about 1.4% v/v to about 17% v/v, about 1.4%v/v to about 16% v/v, about 1.4% v/v to about 15% v/v, about 1.4% v/v toabout 14% v/v, about 1.4% v/v to about 13% v/v, about 1.4% v/v to about12% v/v, about 1.4% v/v to about 11% v/v, about 1.4% v/v to about 10%v/v, about 1.4% v/v to about 9% v/v, about 1.4% v/v to about 8% v/v,about 1.4% v/v to about 7% v/v, about 1.4% v/v to about 6% v/v, about1.4% v/v to about 5% v/v, about 1.4% v/v to about 4.5% v/v, about 1.4%v/v to about 4.0% v/v, about 1.4% v/v to about 3.5% v/v, about 1.4% v/vto about 3.0% v/v, about 1.4% v/v to about 2.5% v/v, about 1.4% v/v toabout 2.2% v/v, about 1.4% v/v to about 2.0% v/v, about 1.4% v/v toabout 1.8% v/v, about 1.4% v/v to about 1.6% v/v, about 1.6% v/v toabout 20% v/v, about 1.6% v/v to about 19% v/v, about 1.6% v/v to about18% v/v, about 1.6% v/v to about 17% v/v, about 1.6% v/v to about 16%v/v, about 1.6% v/v to about 15% v/v, about 1.6% v/v to about 14% v/v,about 1.6% v/v to about 13% v/v, about 1.6% v/v to about 12% v/v, about1.6% v/v to about 11% v/v, about 1.6% v/v to about 10% v/v, about 1.6%v/v to about 9% v/v, about 1.6% v/v to about 8% v/v, about 1.6% v/v toabout 7% v/v, about 1.6% v/v to about 6% v/v, about 1.6% v/v to about 5%v/v, about 1.6% v/v to about 4.5% v/v, about 1.6% v/v to about 4.0% v/v,about 1.6% v/v to about 3.5% v/v, about 1.6% v/v to about 3.0% v/v,about 1.6% v/v to about 2.5% v/v, about 1.6% v/v to about 2.2% v/v,about 1.6% v/v to about 2.0% v/v, about 1.6% v/v to about 1.8% v/v,about 1.8% v/v to about 20% v/v, about 1.8% v/v to about 19% v/v, about1.8% v/v to about 18% v/v, about 1.8% v/v to about 17% v/v, about 1.8%v/v to about 16% v/v, about 1.8% v/v to about 15% v/v, about 1.8% v/v toabout 14% v/v, about 1.8% v/v to about 13% v/v, about 1.8% v/v to about12% v/v, about 1.8% v/v to about 11% v/v, about 1.8% v/v to about 10%v/v, about 1.8% v/v to about 9% v/v, about 1.8% v/v to about 8% v/v,about 1.8% v/v to about 7% v/v, about 1.8% v/v to about 6% v/v, about1.8% v/v to about 5% v/v, about 1.8% v/v to about 4.5% v/v, about 1.8%v/v to about 4.0% v/v, about 1.8% v/v to about 3.5% v/v, about 1.8% v/vto about 3.0% v/v, about 1.8% v/v to about 2.5% v/v, about 1.8% v/v toabout 2.2% v/v, about 1.8% v/v to about 2.0% v/v, about 2.0% v/v toabout 20% v/v, about 2.0% v/v to about 19% v/v, about 2.0% v/v to about18% v/v, about 2.0% v/v to about 17% v/v, about 2.0% v/v to about 16%v/v, about 2.0% v/v to about 15% v/v, about 2.0% v/v to about 14% v/v,about 2.0% v/v to about 13% v/v, about 2.0% v/v to about 12% v/v, about2.0% v/v to about 11% v/v, about 2.0% v/v to about 10% v/v, about 2.0%v/v to about 9% v/v, about 2.0% v/v to about 8% v/v, about 2.0% v/v toabout 7% v/v, about 2.0% v/v to about 6% v/v, about 2.0% v/v to about 5%v/v, about 2.0% v/v to about 4.5% v/v, about 2.0% v/v to about 4.0% v/v,about 2.0% v/v to about 3.5% v/v, about 2.0% v/v to about 3.0% v/v,about 2.0% v/v to about 2.5% v/v, about 2.0% v/v to about 2.2% v/v,about 2.2% v/v to about 20% v/v, about 2.2% v/v to about 19% v/v, about2.2% v/v to about 18% v/v, about 2.2% v/v to about 17% v/v, about 2.2%v/v to about 16% v/v, about 2.2% v/v to about 15% v/v, about 2.2% v/v toabout 14% v/v, about 2.2% v/v to about 13% v/v, about 2.2% v/v to about12% v/v, about 2.2% v/v to about 11% v/v, about 2.2% v/v to about 10%v/v, about 2.2% v/v to about 9% v/v, about 2.2% v/v to about 8% v/v,about 2.2% v/v to about 7% v/v, about 2.2% v/v to about 6% v/v, about2.2% v/v to about 5% v/v, about 2.2% v/v to about 4.5% v/v, about 2.2%v/v to about 4.0% v/v, about 2.2% v/v to about 3.5% v/v, about 2.2% v/vto about 3.0% v/v, about 2.2% v/v to about 2.5% v/v, about 2.5% v/v toabout 20% v/v, about 2.5% v/v to about 19% v/v, about 2.5% v/v to about18% v/v, about 2.5% v/v to about 17% v/v, about 2.5% v/v to about 16%v/v, about 2.5% v/v to about 15% v/v, about 2.5% v/v to about 14% v/v,about 2.5% v/v to about 13% v/v, about 2.5% v/v to about 12% v/v, about2.5% v/v to about 11% v/v, about 2.5% v/v to about 10% v/v, about 2.5%v/v to about 9% v/v, about 2.5% v/v to about 8% v/v, about 2.5% v/v toabout 7% v/v, about 2.5% v/v to about 6% v/v, about 2.5% v/v to about 5%v/v, about 2.5% v/v to about 4.5% v/v, about 2.5% v/v to about 4.0% v/v,about 2.5% v/v to about 3.5% v/v, about 2.5% v/v to about 3.0% v/v,about 3.0% v/v to about 20% v/v, about 3.0% v/v to about 19% v/v, about3.0% v/v to about 18% v/v, about 3.0% v/v to about 17% v/v, about 3.0%v/v to about 16% v/v, about 3.0% v/v to about 15% v/v, about 3.0% v/v toabout 14% v/v, about 3.0% v/v to about 13% v/v, about 3.0% v/v to about12% v/v, about 3.0% v/v to about 11% v/v, about 3.0% v/v to about 10%v/v, about 3.0% v/v to about 9% v/v, about 3.0% v/v to about 8% v/v,about 3.0% v/v to about 7% v/v, about 3.0% v/v to about 6% v/v, about3.0% v/v to about 5% v/v, about 3.0% v/v to about 4.5% v/v, about 3.0%v/v to about 4.0% v/v, about 3.0% v/v to about 3.5% v/v, about 3.5% v/vto about 20% v/v, about 3.5% v/v to about 19% v/v, about 3.5% v/v toabout 18% v/v, about 3.5% v/v to about 17% v/v, about 3.5% v/v to about16% v/v, about 3.5% v/v to about 15% v/v, about 3.5% v/v to about 14%v/v, about 3.5% v/v to about 13% v/v, about 3.5% v/v to about 12% v/v,about 3.5% v/v to about 11% v/v, about 3.5% v/v to about 10% v/v, about3.5% v/v to about 9% v/v, about 3.5% v/v to about 8% v/v, about 3.5% v/vto about 7% v/v, about 3.5% v/v to about 6% v/v, about 3.5% v/v to about5% v/v, about 3.5% v/v to about 4.5% v/v, about 3.5% v/v to about 4.0%v/v, about 4.0% v/v to about 20% v/v, about 4.0% v/v to about 19% v/v,about 4.0% v/v to about 18% v/v, about 4.0% v/v to about 17% v/v, about4.0% v/v to about 16% v/v, about 4.0% v/v to about 15% v/v, about 4.0%v/v to about 14% v/v, about 4.0% v/v to about 13% v/v, about 4.0% v/v toabout 12% v/v, about 4.0% v/v to about 11% v/v, about 4.0% v/v to about10% v/v, about 4.0% v/v to about 9% v/v, about 4.0% v/v to about 8% v/v,about 4.0% v/v to about 7% v/v, about 4.0% v/v to about 6% v/v, about4.0% v/v to about 5% v/v, about 4.0% v/v to about 4.5% v/v, about 4.5%v/v to about 20% v/v, about 4.5% v/v to about 19% v/v, about 4.5% v/v toabout 18% v/v, about 4.5% v/v to about 17% v/v, about 4.5% v/v to about16% v/v, about 4.5% v/v to about 15% v/v, about 4.5% v/v to about 14%v/v, about 4.5% v/v to about 13% v/v, about 4.5% v/v to about 12% v/v,about 4.5% v/v to about 11% v/v, about 4.5% v/v to about 10% v/v, about4.5% v/v to about 9% v/v, about 4.5% v/v to about 8% v/v, about 4.5% v/vto about 7% v/v, about 4.5% v/v to about 6% v/v, about 4.5% v/v to about5% v/v, about 5% v/v to about 20% v/v, about 5% v/v to about 19% v/v,about 5% v/v to about 18% v/v, about 5% v/v to about 17% v/v, about 5%v/v to about 16% v/v, about 5% v/v to about 15% v/v, about 5% v/v toabout 14% v/v, about 5% v/v to about 13% v/v, about 5% v/v to about 12%v/v, about 5% v/v to about 11% v/v, about 5% v/v to about 10% v/v, about5% v/v to about 9% v/v, about 5% v/v to about 8% v/v, about 5% v/v toabout 7% v/v, about 5% v/v to about 6% v/v, about 6% v/v to about 20%v/v, about 6% v/v to about 19% v/v, about 6% v/v to about 18% v/v, about6% v/v to about 17% v/v, about 6% v/v to about 16% v/v, about 6% v/v toabout 15% v/v, about 6% v/v to about 14% v/v, about 6% v/v to about 13%v/v, about 6% v/v to about 12% v/v, about 6% v/v to about 11% v/v, about6% v/v to about 10% v/v, about 6% v/v to about 9% v/v, about 6% v/v toabout 8% v/v, about 6% v/v to about 7% v/v, about 7% v/v to about 20%v/v, about 7% v/v to about 19% v/v, about 7% v/v to about 18% v/v, about7% v/v to about 17% v/v, about 7% v/v to about 16% v/v, about 7% v/v toabout 15% v/v, about 7% v/v to about 14% v/v, about 7% v/v to about 13%v/v, about 7% v/v to about 12% v/v, about 7% v/v to about 11% v/v, about7% v/v to about 10% v/v, about 7% v/v to about 9% v/v, about 7% v/v toabout 8% v/v, about 8% v/v to about 20% v/v, about 8% v/v to about 19%v/v, about 8% v/v to about 18% v/v, about 8% v/v to about 17% v/v, about8% v/v to about 16% v/v, about 8% v/v to about 15% v/v, about 8% v/v toabout 14% v/v, about 8% v/v to about 13% v/v, about 8% v/v to about 12%v/v, about 8% v/v to about 11% v/v, about 8% v/v to about 10% v/v, about8% v/v to about 9% v/v, about 9% v/v to about 20% v/v, about 9% v/v toabout 19% v/v, about 9% v/v to about 18% v/v, about 9% v/v to about 17%v/v, about 9% v/v to about 16% v/v, about 9% v/v to about 15% v/v, about9% v/v to about 14% v/v, about 9% v/v to about 13% v/v, about 9% v/v toabout 12% v/v, about 9% v/v to about 11% v/v, about 9% v/v to about 10%v/v, about 10% v/v to about 20% v/v, about 10% v/v to about 19% v/v,about 10% v/v to about 18% v/v, about 10% v/v to about 17% v/v, about10% v/v to about 16% v/v, about 10% v/v to about 15% v/v, about 10% v/vto about 14% v/v, about 10% v/v to about 13% v/v, about 10% v/v to about12% v/v, about 10% v/v to about 11% v/v, about 11% v/v to about 20% v/v,about 11% v/v to about 19% v/v, about 11% v/v to about 18% v/v, about11% v/v to about 17% v/v, about 11% v/v to about 16% v/v, about 11% v/vto about 15% v/v, about 11% v/v to about 14% v/v, about 11% v/v to about13% v/v, about 11% v/v to about 12% v/v, about 12% v/v to about 20% v/v,about 12% v/v to about 19% v/v, about 12% v/v to about 18% v/v, about12% v/v to about 17% v/v, about 12% v/v to about 16% v/v, about 12% v/vto about 15% v/v, about 12% v/v to about 14% v/v, about 12% v/v to about13% v/v, about 13% v/v to about 20% v/v, about 13% v/v to about 19% v/v,about 13% v/v to about 18% v/v, about 13% v/v to about 17% v/v, about13% v/v to about 16% v/v, about 13% v/v to about 15% v/v, about 13% v/vto about 14% v/v, about 14% v/v to about 20% v/v, about 14% v/v to about19% v/v, about 14% v/v to about 18% v/v, about 14% v/v to about 17% v/v,about 14% v/v to about 16% v/v, about 14% v/v to about 15% v/v, about15% v/v to about 20% v/v, about 15% v/v to about 19% v/v, about 15% v/vto about 18% v/v, about 15% v/v to about 17% v/v, about 15% v/v to about16% v/v, about 16% v/v to about 20% v/v, about 16% v/v to about 19% v/v,about 16% v/v to about 18% v/v, about 16% v/v to about 17% v/v, about17% v/v to about 20% v/v, about 17% v/v to about 19% v/v, about 17% v/vto about 18% v/v, about 18% v/v to about 20% v/v, about 18% v/v to about19% v/v, or about 19% v/v to about 20% v/v.

In some examples of any of the compositions described herein, the liquidcan further include at least one (e.g., two, three, four, or five)antioxidant agent and/or chelator. In some examples of any of thecompositions described herein, the liquid can further include at leastone (e.g., two, three, four, or five) antioxidant agent and/or chelatorin an amount sufficient to ameliorate the loss of binding capacity ofthe chromatography resin after exposure to the dose ofgamma-irradiation.

In some examples of any of the compositions described herein, the liquidcan include at least one (e.g., two, three, four, or five) antioxidantagent selected from the group of: reduced glutathione, reducedthioredoxin, reduced cysteine, a carotenoid, melatonin, lycopene,tocopherol, reduced ubiquinone, ascorbate, bilirubin, uric acid, lipoicacid, a flavonoid, a phenolpropanoid acid, lidocaine, naringenin,fullerene, glucose, mannitol,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, and dimethylmethoxychromanol. In some examples of any of the compositions described herein,the liquid can include at least one (e.g., two, three, or four)antioxidant selected from the group of: mannitol, sodium ascorbate,histidine, and methionine.

In some examples of any of the compositions described herein, the liquidcan contain at least one (e.g., one, two, three, or four) of methionine(or alternatively cysteine or glutathione), sodium ascorbate, histidine,and mannitol. In some examples of any of the compositions describedherein, the liquid can contain methionine (or alternatively cysteine orglutathione), sodium ascorbate, histidine, and mannitol. In someexamples of any of the compositions described herein, the liquid caninclude: (i) between 75 mM and about 125 mM (e.g., between 80 mM andabout 120 mM, between about 85 mM and about 115 mM, between about 90 mMand about 110 mM, or between about 95 mM and about 105 mM) mannitol;(ii) between 75 mM and about 125 mM (e.g., between about 80 mM and about120 mM, between about 85 mM and about 115 mM, between about 90 mM andabout 110 mM, or between about 95 mM and about 105 mM) methionine (oralternatively cysteine or glutathione); (iii) between 75 mM and about125 mM (e.g., between about 80 mM and about 120 mM, between about 85 mMand about 115 mM, between about 90 mM and about 110 mM, or between about95 mM and about 105 mM) sodium ascorbate; (iv) between 75 mM and about125 mM (e.g., between about 80 mM and about 120 mM, between about 85 mMand about 115 mM, between about 90 mM and about 110 mM, or between about95 mM and about 105 mM) histidine; (v) between about 30 mM and about 70mM (e.g., between about 35 mM and about 65 mM, between about 40 mM andabout 60 mM, or between about 45 mM and about 55 mM) methionine (oralternatively cysteine or glutathione) and between about 30 mM and about70 mM (e.g., between about 35 mM and about 65 mM, between about 40 mMand about 60 mM, or between about 45 mM and about 55 mM) histidine; (vi)between about 10 mM and about 50 mM (e.g., between about 15 mM and about45 mM, between about 20 mM and about 40 mM, or between about 25 mM andabout 35 mM) methionine (or alternatively cysteine or glutathione),between about 10 mM and about 50 mM (e.g., between about 15 mM and about45 mM, between about 20 mM and about 40 mM, or between about 25 mM andabout 35 mM) histidine, and between about 10 mM and about 50 mM (e.g.,between about 15 mM and about 45 mM, between about 20 mM and about 40mM, or between about 25 mM and about 35 mM) sodium ascorbate; or (vii)between about 5 mM to about 45 mM (e.g., between about 10 mM and about40 mM, between about 15 mM and about 35 mM, or between about 20 mM andabout 30 mM) sodium ascorbate, between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM) methionine (or alternativelycysteine or glutathione), between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM) mannitol, and between about5 mM and about 45 mM (e.g., between about 10 mM and about 40 mM, betweenabout 15 mM and about 35 mM, or between about 20 mM and about 30 mM)histidine. In some examples of any of the compositions described herein,the liquid can be a buffered solution (e.g., a phosphate bufferedsolution, e.g., a sodium phosphate buffered solution, such as 50 mMsodium phosphate, pH 6.0).

In some embodiments of any of the compositions described herein, theliquid can further include at least one (e.g., two, three, four, orfive) chelator (e.g., at least one chelator selected from the group ofethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonicacid sodium (DMPS), dimercaptosuccinic acid (DMSA), metallothionin, anddesferroxamine).

Also provided herein is a container (e.g., storage vessel, e.g., aplastic container, or a chromatography column) including a composition(e.g., any of the exemplary compositions described herein) including (i)a chromatography resin (e.g., any of the chromatography resins describedherein or known in the art) and (ii) a liquid including at least onealcohol (e.g., any of the exemplary alcohols described herein or knownin the art), wherein the at least one alcohol is present in an amountsufficient to ameliorate the loss of binding capacity of thechromatography resin upon treatment with a dose of gamma-irradiationsufficient to reduce bioburden of the composition. For example, thecontainer (e.g., storage container, e.g., a plastic container, or achromatography column) can have an internal volume of, e.g., at leastabout 1 mL, 5 mL, at least about 10 mL, at least about 20 mL, at leastabout 30 mL, at least about 40 mL, at least about 50 mL, at least about60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL,at least about 100 mL, at least about 110 mL, at least about 120 mL, atleast about 130 mL, at least about 140 mL, at least about 150 mL, atleast about 160 mL, at least about 170 mL, at least about 180 mL, atleast about 190 mL, at least about 200 mL, at least about 210 mL, atleast about 220 mL, at least about 230 mL, at least about 240 mL, atleast about 250 mL, at least 300 mL, at least 350 mL, at least 400 mL,or at least 500 mL. For example, a container can have an internal volumeof, e.g., between about 1 mL and about 500 mL, between about 1 mL andabout 50 mL, between about 5 mL and about 500 mL, between about 5 mL andabout 400 mL, between about 5 mL and about 350 mL, between about 5 mLand about 300 mL, between about 5 mL and about 250 mL, between about 5mL and about 200 mL, between about 5 mL and about 150 mL, between about5 mL and about 100 mL, or between about 5 mL and about 50 mL. In someexamples, the chromatography resin in the container is a slurry ofsedimented chromatography resin in the liquid. In some examples, thecontainer includes a packed chromatography resin (e.g., packed in theliquid).

In any of the compositions provided herein, the liquid can furthercontain at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antioxidantagent and/or at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10)chelator. Any of the antioxidant agent(s) that can be included in any ofthe compositions provided herein can have the ability to quench one ormore of the following reactive oxygen and/or nitrogen species: hydroxylradical, carbonate radical, superoxide anion, peroxyl radical,peroxynitrite, nitrogen dioxide, and nitric oxide. Non-limiting examplesof antioxidant agents that can be included in any of the compositionsprovided herein include: reduced glutathione, reduced thioredoxin,reduced cysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol. Additional non-limiting examples ofantioxidant agents include antioxidant enzymes (e.g., superoxidedismutase, glutathione peroxidase, glutathione reductase, catalase, andthioredoxin reductase). Additional examples of antioxidant agents thatcan be included in any of the compositions provided herein includemannitol, sodium ascorbate, methionine, and histidine. Further examplesof antioxidant agents include cysteine, taurine,mercaptopropionylglycine, N-acetylcysteine, garlic oil, diallylsulfide,dihydrolipoic acid, and diallyltrisulfide. Some embodiments that includean antioxidant enzyme as an antioxidant agent can further include one ormore substrate(s) for the enzyme. An antioxidant agent can be identifiedusing a number of methods known in the art including, for example, spintrapping, redox sensitive dyes, and chemiluminescence assays.

Any of the chelator(s) that can be included in any of the compositionsprovided herein can have the ability to bind a redox active metal (e.g.,Cu²⁺ and Fe²⁺) with high affinity (e.g., about or less than 1 μM, aboutor less than 800 nM, about or less than 700 nM, about or less than 600nM, about or less than 500 nM, about or less than 400 nM, about or lessthan 300 nM, about or less than 250 nM, about or less than 200 nM, aboutor less than 150 nM, about or less than 100 nM, about or less than 80nM, about or less than 60 nM, about or less than 40 nM, about or lessthan 20 nM, or about or less than 1 nm). Non-limiting examples ofchelator(s) that can be included in any of the compositions providedherein include ethylenediaminetetraacetic acid (EDTA),2,3-dimercapto-1-propanesulfonic acid sodium (DMPS), dimercaptosuccinicacid (DMSA), metallothionin, and desferroxamine.

The concentration of each of the chelator(s) and/or antioxidant(s) thatcan be included in any of the compositions provided herein can bebetween about 0.1 mM and about 150 mM (e.g., between about 0.1 mM andabout 150 mM, between about 0.1 mM and about 125 mM, between about 0.1mM and about 100 mM, between about 0.1 mM and about 80 mM, between about0.1 mM and about 60 mM, between about 0.1 mM and about 50 mM, betweenabout 0.1 mM and about 40 mM, between about 0.1 mM and about 30 mM,between about 0.1 mM and about 25 mM, between about 0.1 mM and about 20mM, between about 0.1 mM and about 10 mM, between about 0.1 mM and about5.0 mM, between about 0.5 mM and about 150 mM, between about 0.5 mM andabout 100 mM, between about 0.5 mM and about 50 mM, between about 0.5 mMand about 25 mM, between about 0.5 mM and about 15 mM, between about 0.5mM and about 10 mM, between about 0.5 mM and about 5 mM, between about 1mM and about 125 mM, between about 1 mM and about 120 mM, between about1 mM and about 100 mM, between about 1 mM and about 80 mM, between about1 mM and about 60 mM, between about 1 mM and about 50 mM, between about1 mM and about 40 mM, between about 1 mM and about 30 mM, between about1 mM and about 25 mM, between about 5 mM and about 150 mM, between about5 mM and about 125 mM, between about 5 mM and about 100 mM, betweenabout 5 mM and about 80 mM, between about 5 mM and about 60 mM, betweenabout 5 mM and about 50 mM, between about 5 mM and about 40 mM, betweenabout 5 mM and about 30 mM, between about 5 mM and about 25 mM, betweenabout 10 mM and about 150 mM, between about 10 mM and about 125 mM,between about 1 mM and about 100 mM, between about 10 mM and about 80mM, between about 10 mM and about 60 mM, between about 10 mM and about50 mM, between about 10 mM and about 40 mM, between about 10 mM andabout 30 mM, between about 10 mM and about 25 mM, between about 20 mMand about 150 mM, between about 20 mM and about 125 mM, between about 20mM and about 100 mM, between about 20 mM and about 80 mM, between about20 mM and about 60 mM, between about 20 mM and about 50 mM, betweenabout 20 mM and about 40 mM, between about 20 mM and about 30 mM,between about 30 mM and about 150 mM, between about 30 mM and about 125mM, between about 30 mM and about 100 mM, between about 30 mM and about80 mM, between about 30 mM and about 60 mM, between about 30 mM andabout 50 mM, between about 30 mM and about 40 mM, between about 40 mMand about 150 mM, between about 40 mM and about 125 mM, between about 40mM and about 100 mM, between about 40 mM and about 90 mM, between about40 mM and about 80 mM, between about 40 mM and about 70 mM, betweenabout 40 mM and about 60 mM, between about 50 mM and about 150 mM,between about 50 mM and about 125 mM, between about 50 mM and about 100mM, between about 50 mM and about 80 mM, between about 50 mM and about60 mM, between about 80 mM and about 150 mM, between about 80 mM andabout 125 mM, between about 80 mM and about 100 mM, between about 100 mMand about 150 mM, or between about 100 mM and about 125 mM).

In some examples, the compositions provided herein can contain one ormore of 5 mM to about 150 mM mannitol (e.g., between about 10 mM andabout 150 mM, between about 20 mM and about 150 mM, between about 30 mMand about 150 mM, between about 40 mM and about 150 mM, between about 50mM and about 150 mM, between about 60 mM and about 140 mM, between about70 mM and about 130 mM, between about 80 mM and about 120 mM, betweenabout 90 mM and about 110 mM, between about 95 mM and about 105 mM,between about 5 mM and about 50 mM, between about 5 mM and about 45 mM,between about 5 mM and about 40 mM, between about 5 mM and about 35 mM,between about 10 mM and about 35 mM, between about 15 mM and about 35mM, or between about 20 mM and about 30 mM mannitol); 5 mM to about 150mM (e.g., between about 10 mM and about 150 mM, between about 20 mM andabout 150 mM, between about 30 mM and about 150 mM, between about 40 mMand about 150 mM, between about 50 mM and about 150 mM, between about 60mM and about 140 mM, between about 70 mM and about 130 mM, between about80 mM and about 120 mM, between about 90 mM and about 110 mM, betweenabout 95 mM and about 105 mM, between about 30 mM and about 70 mM,between about 35 mM and about 65 mM, between about 40 mM and about 60mM, between about 45 mM and about 55 mM, between about 20 mM and about50 mM, between about 25 mM and about 45 mM, between about 30 mM andabout 40 mM, between about 30 mM and about 35 mM, between about 5 mM andabout 45 mM, between about 10 mM and about 40 mM, between about 15 mMand about 35 mM, between about 20 mM and about 30 mM, or between about20 mM and about 25 mM) methionine (or alternatively cysteine orglutathione); 5 mM to 150 mM sodium ascorbate (e.g., between about 10 mMand about 150 mM, between about 20 mM and about 150 mM, between about 30mM and about 150 mM, between about 40 mM and about 150 mM, between about50 mM and about 150 mM, between about 60 mM and about 140 mM, betweenabout 70 mM and about 130 mM, between about 80 mM and about 120 mM,between about 90 mM and about 110 mM, between about 95 mM and about 105mM, between about 10 mM and about 50 mM, between about 15 mM and about45 mM, between about 20 mM and about 40 mM, between about 25 mM andabout 35 mM, between about 30 mM and about 35 mM, between about 5 mM andabout 45 mM, between about 10 mM and about 40 mM, between about 15 mMand about 35 mM, between about 20 mM and about 30 mM, between about 20mM and about 25 mM sodium ascorbate); and 5 mM to about 150 mM (e.g.,between about 10 mM and about 150 mM, between about 20 mM and about 150mM, between about 30 mM and about 150 mM, between about 40 mM and about150 mM, between about 50 mM and about 150 mM, between about 60 mM andabout 140 mM, between about 70 mM and about 130 mM, between about 80 mMand about 120 mM, between about 90 mM and about 110 mM, between about 95mM and about 105 mM, between about 30 mM and about 70 mM, between about35 mM and about 65 mM, between about 40 mM and about 60 mM, betweenabout 45 mM and about 55 mM, between about 20 mM and about 50 mM,between about 25 mM and about 45 mM, between about 30 mM and about 40mM, between about 30 mM and about 35 mM, between about 5 mM and about 45mM, between about 10 mM and about 40 mM, between about 15 mM and about35 mM, between about 20 mM and about 30 mM, or between about 20 mM andabout 25 mM) histidine.

Non-limiting examples of any of the compositions can contain (i) betweenabout 75 mM and about 125 mM (e.g., between about 80 mM and about 120mM, between about 85 mM and about 115 mM, between about 90 mM and about110 mM, or between about 95 mM and about 105 mM) mannitol (e.g., in abuffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0); (ii) between about 75 mM and about 125 mM (e.g.,between about 80 mM and about 120 mM, between about 85 mM and about 115mM, between about 90 mM and about 110 mM, or between about 95 mM andabout 105 mM) methionine (or alternatively cysteine or glutathione)(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (iii) between about 75 mM and about 125 mM(e.g., between about 80 mM and about 120 mM, between about 85 mM andabout 115 mM, between about 90 mM and about 110 mM, or between about 95mM and about 105 mM) sodium ascorbate (e.g., in a buffered solution,e.g., a phosphate buffer, such as 50 mM sodium phosphate, pH 6.0); (iv)between about 75 mM and about 125 mM (e.g., between about 80 mM andabout 120 mM, between about 85 mM and about 115 mM, between about 90 mMand about 110 mM, or between about 95 mM and about 105 mM) histidine(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (v) between about 30 mM and about 70 mM(e.g., between about 35 mM and about 65 mM, between about 40 mM andabout 60 mM, or between about 45 mM and about 55 mM) methionine (oralternatively cysteine or glutathione) and between about 30 mM and about70 mM (e.g., between about 35 mM and about 65 mM, between about 40 mMand about 60 mM, or between about 45 mM and about 55 mM) histidine(e.g., in a buffered solution, e.g., a phosphate buffer, such as 50 mMsodium phosphate, pH 6.0); (vi) between about 10 mM and about 50 mM(e.g., between about 15 mM and about 45 mM, between about 20 mM andabout 40 mM, between about 25 mM to about 35 mM, or between about 30 mMand about 35 mM) methionine (or alternatively cysteine or glutathione),between about 10 mM and about 50 mM (e.g., between about 15 mM and about45 mM, between about 20 mM and about 40 mM, between about 25 mM to about35 mM, or between about 30 mM and about 35 mM) histidine, and betweenabout 10 mM and about 50 mM (e.g., between about 15 mM and about 45 mM,between about 20 mM and about 40 mM, between about 25 mM to about 35 mM,or between about 30 mM to about 35 mM) sodium ascorbate (e.g., in abuffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0); or (vii) between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, between about 20 mM and about 30 mM, or between about 23 mM andabout 27 mM) sodium ascorbate, between about 5 mM and about 45 mM (e.g.,between about 10 mM and about 40 mM, between about 15 mM and about 35mM, between about 20 mM and about 30 mM, or between about 23 mM andabout 27 mM) methionine (or alternatively cysteine or glutathione),between about 5 mM and about 45 mM (e.g., between about 10 mM and about40 mM, between about 15 mM and about 35 mM, between about 20 mM andabout 30 mM, or between about 23 mM and about 27 mM) mannitol, andbetween about 5 mM and about 45 mM (e.g., between about 10 mM and about40 mM, between about 15 mM and about 35 mM, between about 20 mM andabout 30 mM, or between about 23 mM and about 27 mM) histidine (e.g., ina buffered solution, e.g., a phosphate buffer, such as 50 mM sodiumphosphate, pH 6.0).

Non-limiting doses of gamma-irradiation sufficient to reduce bioburdenof any of the compositions provided herein are described below.Additional doses of gamma-irradiation sufficient to reduce bioburden ofany of the compositions provided herein are known in the art. Forexample, any of the compositions described herein can begamma-irradiated at any of the doses, at any of the rates ofgamma-irradiation, and/or at any of the temperatures for performinggamma-irradiation described herein (in any combination). The bioburdenof a composition can be determined, e.g., by taking a sample from thecomposition that would contain self-replicating biologicalcontaminant(s) present in the composition, e.g., by stomaching,ultrasonicating, shaking, vortex mixing, flushing, blending, orswabbing, and qualitating or quantifying the level of self-replicatingbiological contaminant(s) present in the sample (e.g., by placing thesample in a growth medium that would allow the biological contaminant toself-replicate, e.g., plating the sample on a petri dish, or running thesample through a membrane).

The amount of the at least one alcohol, the at least one antioxidantagent and/or the at least one chelator sufficient to ameliorate the lossof binding capacity of the chromatography resin upon treatment with adose of gamma-irradiation sufficient to reduce bioburden of thecomposition can be determined, e.g., using methods described in theExamples. For example, the level of reduction in the binding capacity ofa chromatography resin treated with gamma-irradiation in the presence ofan amount of the at least one alcohol (and optionally, further in thepresence of at least one antioxidant agent and/or chelator) can becompared to the level of reduction in the binding capacity of thechromatography resin treated with the same dose of gamma-irradation inthe absence of the at least one alcohol (and optionally, at least oneantioxidant agent and/or chelator), where a decrease in the level ofreduction in the binding capacity of the chromatography resingamma-irradiated in the presence of the at least one alcohol (andoptionally, further in the presence of at least one antioxidant agentand/or chelator) as compared to the chromatography resingamma-irradiated in the absence of the at least one alcohol (andoptionally, at least one antioxidant agent and/or chelator), indicatesthat the at least one alcohol (and optionally, the antioxidant agentand/or chelator) was present in an amount sufficient to ameliorate theloss of binding capacity of the chromatography resin upon treatment withgamma-irradiation. Exemplary methods for determining the bindingcapacity of a chromatography resin are described in the Examples.Additional examples of methods for determining the binding capacity of achromatography resin are known in the art.

Methods of Reducing Bioburden of a Chromatography Resin

Provided herein are methods of reducing bioburden of a chromatographyresin. These methods include a step of exposing a container including acomposition including (i) a chromatography resin and (ii) a liquidincluding at least one alcohol (e.g., any of the exemplary compositionsincluding a chromatography resin and a liquid including at least onealcohol described herein), to a dose of gamma-irradiation sufficient toreduce the bioburden of the container and the chromatography resin,where the at least one alcohol is present in an amount sufficient toameliorate the loss of binding capacity of the chromatography resinafter (or upon) exposure to the dose of gamma-irradiation.

Also provided are methods of reducing bioburden of a chromatographyresin that include exposing a container including a compositionincluding (i) a chromatography resin and (ii) a liquid including atleast one alcohol in an amount sufficient to ameliorate loss of bindingcapacity of the chromatography resin after/upon exposure togamma-irradiation (e.g., any of the compositions including achromatography resin and a liquid including at least one alcoholdescribed herein), to gamma-irradiation at a rate of between about 0.1kGy/hour to about 6 kGy/hour (e.g., between about 0.1 kGy/hour to about5.5 kGy/hour, between about 0.1 kGy/hour to about 5.0 kGy/hour, betweenabout 0.1 kGy/hour to about 4.5 kGy/hour, between about 0.1 kGy/hour toabout 4.0 kGy/hour, between about 0.1 kGy/hour to about 3.5 kGy/hour,between about 0.1 kGy/hour to about 3.0 kGy/hour, between about 0.1kGy/hour to about 2.5 kGy/hour, between about 0.1 kGy/hour to about 2.0kGy/hour, between about 0.1 kGy/hour to about 1.5 kGy/hour, betweenabout 0.1 kGy/hour to about 1.0 kGy/hour, between about 0.5 kGy/hour toabout 6 kGy/hour, between about 0.5 kGy/hour to about 5.5 kGy/hour,between about 0.5 kGy/hour to about 5.0 kGy/hour, between about 0.5kGy/hour to about 4.5 kGy/hour, between about 0.5 kGy/hour to about 4.0kGy/hour, between about 0.5 kGy/hour to about 3.5 kGy/hour, betweenabout 0.5 kGy/hour to about 3.0 kGy/hour, between about 0.5 kGy/hour toabout 2.5 kGy/hour, between about 0.5 kGy/hour to about 2.0 kGy/hour)and/or at a temperature between about 4° C. to about 25° C. (e.g.,between about 4° C. to about 20° C., between about 4° C. to about 15°C., between about 4° C. to about 10° C., between about 10° C. to about25° C., between about 10° C. to about 20° C., between about 10° C. toabout 15° C., or between about 15° C. to about 25° C.) for a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the chromatography resin.

Some embodiments of any of these methods can include, before and/orafter the exposing step, storing the container or the compositionincluding the chromatography resin and the liquid including at least onealcohol (e.g., any of the containers or any of the compositionsincluding the chromatography resin and the liquid including at least onealcohol described herein) for a period of about 1 hour to about 1 year,about 1 hour to about 11 months, about 1 hour to about 10 months, about1 hour to about 9 months, about 1 hour to about 8 months, about 1 hourto about 7 months, about 1 hour to about 6 months, about 1 hour to about5 months, about 1 hour to about 4 months, about 1 hour to about 3months, about 1 hour to about 2 months, about 1 hour to about 1 month,about 1 hour to about 2 weeks, about 1 hour to about 1 week, about 1hour to about 5 days, about 1 hour to about 2 days, about 1 hour toabout 1 day, about 1 hour to about 12 hours, about 1 hour to about 6hours, about 6 hours to about 1 year, about 6 hours to about 11 months,about 6 hours to about 10 months, about 6 hours to about 9 months, about6 hours to about 8 months, about 6 hours to about 7 months, about 6hours to about 6 months, about 6 hours to about 5 months, about 6 hoursto about 4 months, about 6 hours to about 3 months, about 6 hours toabout 2 months, about 6 hours to about 1 month, about 6 hours to about 2weeks, about 6 hours to about 1 week, about 6 hours to about 5 days,about 6 hours to about 2 days, about 6 hours to about 1 day, about 6hours to about 12 hours, about 12 hours to about 1 year, about 12 hoursto about 11 months, about 12 hours to about 10 months, about 12 hours toabout 9 months, about 12 hours to about 8 months, about 12 hours toabout 7 months, about 12 hours to about 6 months, about 12 hours toabout 5 months, about 12 hours to about 4 months, about 12 hours toabout 3 months, about 12 hours to about 2 months, about 12 hours toabout 1 month, about 12 hours to about 2 weeks, about 12 hours to about1 week, about 12 hours to about 5 days, about 12 hours to about 2 days,about 12 hours to about 1 day, about 1 day to about 1 year, about 1 dayto about 11 months, about 1 day to about 10 months, about 1 day to about9 months, about 1 day to about 8 months, about 1 day to about 7 months,about 1 day to about 6 months, about 1 day to about 5 months, about 1day to about 4 months, about 1 day to about 3 months, about 1 day toabout 2 months, about 1 day to about 1 month, about 1 day to about 2weeks, about 1 day to about 1 week, about 1 day to about 5 days, about 1day to about 2 days, about 2 days to about 1 year, about 2 days to about11 months, about 2 days to about 10 months, about 2 days to about 9months, about 2 days to about 8 months, about 2 days to about 7 months,about 2 days to about 6 months, about 2 days to about 5 months, about 2days to about 4 months, about 2 days to about 3 months, about 2 days toabout 2 months, about 2 days to about 1 month, about 2 days to about 2weeks, about 2 days to about 1 week, about 2 days to about 5 days, about5 days to about 1 year, about 5 days to about 11 months, about 5 days toabout 10 months, about 5 days to about 9 months, about 5 days to about 8months, about 5 days to about 7 months, about 5 days to about 6 months,about 5 days to about 5 months, about 5 days to about 4 months, about 5days to about 3 months, about 5 days to about 2 months, about 5 days toabout 1 month, about 5 days to about 2 weeks, about 5 days to about 1week, about 1 week to about 1 year, about 1 week to about 11 months,about 1 week to about 10 months, about 1 week to about 9 months, about 1week to about 8 months, about 1 week to about 7 months, about 1 week toabout 6 months, about 1 week to about 5 months, about 1 week to about 4months, about 1 week to about 3 months, about 1 week to about 2 months,about 1 week to about 1 month, about 1 week to about 2 weeks, about 2weeks to about 1 year, about 2 weeks to about 11 months, about 2 weeksto about 10 months, about 2 weeks to about 9 months, about 2 weeks toabout 8 months, about 2 weeks to about 7 months, about 2 weeks to about6 months, about 2 weeks to about 5 months, about 2 weeks to about 4months, about 2 weeks to about 3 months, about 2 weeks to about 2months, about 2 weeks to about 1 month, about 1 month to about 1 year,about 1 month to about 11 months, about 1 month to about 10 months,about 1 month to about 9 months, about 1 month to about 8 months, about1 month to about 7 months, about 1 month to about 6 months, about 1month to about 5 months, about 1 month to about 4 months, about 1 monthto about 3 months, about 1 month to about 2 months, about 2 months toabout 1 year, about 2 months to about 11 months, about 2 months to about10 months, about 2 months to about 9 months, about 2 months to about 8months, about 2 months to about 7 months, about 2 months to about 6months, about 2 months to about 5 months, about 2 months to about 4months, about 2 months to about 3 months, about 3 months to about 1year, about 3 months to about 11 months, about 3 months to about 10months, about 3 months to about 9 months, about 3 months to about 8months, about 3 months to about 7 months, about 3 months to about 6months, about 3 months to about 5 months, about 3 months to about 4months, about 4 months to about 1 year, about 4 months to about 11months, about 4 months to about 10 months, about 4 months to about 9months, about 4 months to about 8 months, about 4 months to about 7months, about 4 months to about 6 months, about 4 months to about 5months, about 5 months to about 1 year, about 5 months to about 11months, about 5 months to about 10 months, about 5 months to about 9months, about 5 months to about 8 months, about 5 months to about 7months, about 5 months to about 6 months, about 6 months to about 1year, about 6 months to about 11 months, about 6 months to about 10months, about 6 months to about 9 months, about 6 months to about 8months, about 6 months to about 7 months, about 7 months to about 1year, about 7 months to about 11 months, about 7 months to about 10months, about 7 months to about 9 months, about 7 months to about 8months, about 8 months to about 1 year, about 8 months to about 11months, about 8 months to about 10 months, about 8 months to about 9months, about 9 months to about 1 year, about 9 months to about 11months, about 9 months to about 10 months, about 10 months to about 1year, about 10 months to about 11 months, or about 11 months to about 1year, e.g., at a temperature of about 4° C. to about 40° C., about 4° C.to about 35° C., about 4° C. to about 30° C., about 4° C. to about 28°C., about 4° C. to about 26° C., about 4° C. to about 24° C., about 4°C. to about 22° C., about 4° C. to about 20° C., about 4° C. to about18° C., about 4° C. to about 16° C., about 4° C. to about 14° C., about4° C. to about 12° C., about 4° C. to about 10° C., about 4° C. to about8° C., about 4° C. to about 6° C., about 6° C. to about 40° C., about 6°C. to about 35° C., about 6° C. to about 30° C., about 6° C. to about28° C., about 6° C. to about 26° C., about 6° C. to about 24° C., about6° C. to about 22° C., about 6° C. to about 20° C., about 6° C. to about18° C., about 6° C. to about 16° C., about 6° C. to about 14° C., about6° C. to about 12° C., about 6° C. to about 10° C., about 6° C. to about8° C., about 8° C. to about 40° C., about 8° C. to about 35° C., about8° C. to about 30° C., about 8° C. to about 28° C., about 8° C. to about26° C., about 8° C. to about 24° C., about 8° C. to about 22° C., about8° C. to about 20° C., about 8° C. to about 18° C., about 8° C. to about16° C., about 8° C. to about 14° C., about 8° C. to about 12° C., about8° C. to about 10° C., about 10° C. to about 40° C., about 10° C. toabout 35° C., about 10° C. to about 30° C., about 10° C. to about 28°C., about 10° C. to about 26° C., about 10° C. to about 24° C., about10° C. to about 22° C., about 10° C. to about 20° C., about 10° C. toabout 18° C., about 10° C. to about 16° C., about 10° C. to about 14°C., about 10° C. to about 12° C., about 12° C. to about 40° C., about12° C. to about 35° C., about 12° C. to about 30° C., about 12° C. toabout 28° C., about 12° C. to about 26° C., about 12° C. to about 24°C., about 12° C. to about 22° C., about 12° C. to about 20° C., about12° C. to about 18° C., about 12° C. to about 16° C., about 12° C. toabout 14° C., about 14° C. to about 40° C., about 14° C. to about 35°C., about 14° C. to about 30° C., about 14° C. to about 28° C., about14° C. to about 26° C., about 14° C. to about 24° C., about 14° C. toabout 22° C., about 14° C. to about 20° C., about 14° C. to about 18°C., about 14° C. to about 16° C., about 16° C. to about 40° C., about16° C. to about 35° C., about 16° C. to about 30° C., about 16° C. toabout 28° C., about 16° C. to about 26° C., about 16° C. to about 24°C., about 16° C. to about 22° C., about 16° C. to about 20° C., about16° C. to about 18° C., about 18° C. to about 40° C., about 18° C. toabout 35° C., about 18° C. to about 30° C., about 18° C. to about 28°C., about 18° C. to about 26° C., about 18° C. to about 24° C., about18° C. to about 22° C., about 18° C. to about 20° C., about 20° C. toabout 40° C., about 20° C. to about 35° C., about 20° C. to about 30°C., about 20° C. to about 28° C., about 20° C. to about 26° C., about20° C. to about 24° C., about 20° C. to about 22° C., about 22° C. toabout 40° C., about 22° C. to about 35° C., about 22° C. to about 30°C., about 22° C. to about 28° C., about 22° C. to about 26° C., about22° C. to about 24° C., about 24° C. to about 40° C., about 24° C. toabout 35° C., about 24° C. to about 30° C., about 24° C. to about 28°C., about 24° C. to about 26° C., about 26° C. to about 40° C., about26° C. to about 35° C., about 26° C. to about 30° C., about 26° C. toabout 28° C., about 28° C. to about 40° C., about 28° C. to about 35°C., about 28° C. to about 30° C., about 30° C. to about 40° C., about30° C. to about 35° C., or about 35° C. to about 40° C.

In the methods described in this paragraph, the level of bindingcapacity of the gamma-irradiated chromagraphy resin produced by thesemethods is greater than the level of binding capacity of agamma-irradiated chromagraphy resin gamma-irradiated at one or both of arate of greater than 6.1 kGy/hour and/or at a temperature greater than25° C.

A chromatography resin can be exposed to gamma-irradiation using methodsknown in the art. For example, an isotope such as Cobalt-60 orCaesium-137 can be used as the source of gamma-rays. The chromatographyresin can be exposed to gamma-irradiation at a temperature of aboutbetween about −25° C. and about 0° C., inclusive, or between about 0° C.and about 25° C., inclusive. The chromagraphy resin can be exposed to adose of gamma-irradiation of between about 0.1 kGy to about 100 kGy,between about 1 kGy to about 100 kGy, between about 1 kGy to about 90kGy, between about 1 kGy to about 80 kGy, between about 1 kGy to about70 kGy, between about 1 kGy to about 65 kGy, between about 5 kGy toabout 65 kGy, between about 10 kGy to about 60 kGy, between about 10 kGyto about 55 kGy, between about 10 kGy to about 50 kGy, between about 10kGy to about 45 kGy, between about 10 kGy to about 40 kGy, between about10 kGy to about 35 kGy, between about 10 kGy to about 30 kGy, betweenabout 15 kGy to about 50 kGy, between about 15 kGy to about 45 kGy,between about 15 kGy to about 40 kGy, between about 15 kGy to about 35kGy, between about 20 kGy to about 30 kGy, or between about 23 kGy toabout 27 kGy. The chromatography resin can be exposed togamma-irradiation in a dose sufficient to result in a sterilityassurance level of the chromatography resin of about or less than1×10⁻⁶, about or less than 1×10⁻⁷, about or less than 10×10⁻⁸, about orless than 1×10⁻¹¹, or about or less than 1×10⁻¹², or between about1×10⁻⁶ and about 1×10⁻¹², between about 1×10⁻⁶ and about 1×10⁻¹¹,between about 1×10⁻⁶ and about 1×10⁻¹⁰, between about 1×10⁻⁶ and about1×10⁻⁹, between about 1×10⁻⁶ and about 1×10⁻⁸, between 1×10⁻⁶ and about1×10⁻⁷, between about 1×10⁻⁷ and about 1×10⁻¹² between about 1×10⁻⁷ andabout 1×10⁻¹¹, between about 1×10⁻⁷ and about 1×10⁻¹⁰ between about1×10⁻⁷ and about 1×10⁻⁹, between about 1×10⁻⁷ and about 1×10⁻⁸, betweenabout 1×10⁻⁸ and about 1×10⁻¹², between about 1×10⁻⁸ and about 1×10⁻¹¹,between about 1×10⁻⁸ and about 1×10⁻¹⁰, or between about 1×10⁻⁸ andabout 1×10⁻⁹.

A dose of gamma-irradiation sufficient to reduce the bioburden of achromatography resin can be determined using methods known in the art.For example, the bioburden level of a chromatography resin treated witha dose of gamma-irradiation can be compared to the bioburden level of anuntreated (e.g., a control, non-gamma-irradiated) chromatography resin,and a decrease in the level of bioburden in the gamma-irradiatedchromatography resin as compared to the untreated chromatography resinindicates that the dose of gamma-irradiation is sufficient to reduce thebioburden of a chromatography resin. Exemplary methods for determiningthe level of bioburden in a composition (e.g., a chromatography resin)are described herein. Additional methods for determining the level ofbioburden in a composition (e.g., a chromatography resin) are known inthe art.

The chromatography resin in any of these methods can be an anionicexchange chromatography resin, cationic exchange chromatography resin,size exclusion chromatography resin, hydrophobic interactionchromatography resin, or an affinity chromatography resin, or anycombination thereof. Non-limiting examples of an affinity chromatographyresin can include a protein or peptide ligand (e.g., between about 5amino acids to about 100 amino acids, between about 5 amino acids toabout 90 amino acids, between about 5 amino acids and about 80 aminoacids, between about 5 amino acids and about 70 amino acids, betweenabout 5 amino acids and about 60 amino acids, between about 5 aminoacids and about 50 amino acids, between about 5 amino acids and about 40amino acids, between about 5 amino acids and about 30 amino acids,between about 5 amino acids and about 25 amino acids, or between about 5amino acids and about 20 amino acids), a small molecule substrate orcofactor of an enzyme, an aptamer, an inhibitor (e.g., competitiveprotein inhibitor) or a metal. In some embodiments, the affinitychromatography resin includes a protein ligand (e.g., protein A).Additional examples of affinity chromatography resin include a cofactorligand, a substrate ligand, a metal ligand, a product ligand, or anaptamer ligand. In some examples, the chromatography resin is a biomodalchromatography resin (e.g., anionic exchange chromatography resin andhydrophobic interaction chromatography resin). The chromatography resincan be an anionic exchange chromatography resin (e.g., an anionicexchange chromatography resin including N-benzyl-N-methyl-ethanolaminegroups).

The container that includes a chromatography resin can be a plasticcontainer (e.g., a cylindrical tube, a sealed or clamped box, or asealed bag). Non-limiting examples of containers used in these methodsinclude a storage vessel or a chromatography column. For example, thecomposition including (i) the chromatography resin and (ii) the liquidincluding at least one alcohol (e.g., any of the exemplary compositionsdescribed herein) can be present in a sealed container (e.g., a slurryin a sealed container or a packed chromatography resin in a sealedcontainer (e.g., chromatography column)). A container used in themethods described herein can be a disposable chromatography column. Insome embodiments, the container used in the methods described herein isa disposable chromatography column placed in a blister pack. Thecontainer (e.g., storage vessel or chromatography column) can have aninternal total volume of between about 1 mL to about 1 L (e.g., betweenabout 1 mL and about 900 mL, between about 1 mL and about 800 mL,between about 1 mL and about 700 mL, between about 1 mL and about 600mL, between about 1 mL and about 500 mL, between about 1 mL and about450 mL, between about 1 mL and about 400 mL, between about 1 mL andabout 350 mL, between about 1 mL and about 300 mL, between about 1 mLand about 250 mL, between about 1 mL and about 200 mL, between about 1mL and about 150 mL, between about 1 mL and about 100 mL, between about1 mL and about 75 mL, between about 1 mL and about 50 mL, between about1 mL and about 40 mL, between about 1 mL and about 30 mL, or betweenabout 1 mL and about 20 mL).

The composition containing (i) a chromatography resin and (ii) a liquidincluding the at least one alcohol (e.g., any of the exemplarycompositions described herein) included in the container can be presentas a wetted or moist solid mixture. For example, the container caninclude a slurry of a sedimented chromatography resin in the liquid. Insome embodiments, the container can include a packed chromatographyresin. For example, the container including the composition including(i) the chromatography resin and (ii) a liquid including the at leastone alcohol (e.g., any of the compositions described herein) is a packedchromatography column (e.g., where the resin is packed in the liquidincluding the at least one alcohol). Some embodiments include, prior toexposing, disposing the composition containing (i) the chromatographyresin and (ii) the liquid including the at least one alcohol (e.g., anyof the compositions described herein) into the container.

Any of the alcohols, antioxidant agents, and/or chelators describedherein can be used in any combination, using any combination of theexemplary concentrations described herein. For example, the liquid caninclude at least one alcohol selected from the group of: benzyl alcohol,cyclohexanol, isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol,propan-2-ol, propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol,2-phenyl ethanol, sec-phenyl ethanol, 3-phenyl-1-propanol,1-phenyl-1-propanol, 2-phenyl-1-propanol, 2-phenyl-2-propanol,1-phenyl-2-butanol, 2-phenyl-1-butanol, 3-phenyl-1-butanol,4-phenyl-2-butanol, dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and4-phenyl-1-butanol. In some examples, the liquid can further include atleast one antioxidant agent (e.g., at least one antioxidant agentselected from the group of reduced glutathione, reduced thioredoxin,reduced cysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol) and/or at least one chelator (e.g., at leastone chelator selected from the group of EDTA, DMPS, DMSA,metallothionin, and desferroxamine).

Exemplary methods for determining/identifying amount(s) of the at leastone alcohol, at least one antioxidant agent, and/or at least onechelator sufficient to ameliorate the loss of binding capacity of thechromatography resin upon treatment with a dose of gamma-irradiationsufficient to reduce bioburden of the composition are described herein.Additional methods for determining/identifying amount(s) of the at leastone antioxidant agent and/or chelator sufficient to ameliorate the lossof binding capacity of the chromatography resin upon treatment with adose of gamma-irradiation sufficient to reduce bioburden of thecomposition are known in the art.

Also provided herein are a reduced bioburden chromatography resinproduced by any of the methods described herein (e.g., a reducedbioburden chromatography resin provided in a storage container, e.g., asealed storage container). The reduced bioburden chromatography resingenerated using any of the methods described herein can have a sterilityassurance level of about or less than 1×10⁻⁶, about or less than 1×10⁻⁷,about or less than 10×10⁻⁸, about or less than 1×10⁻¹¹, or about or lessthan 1×10⁻¹², or between about 1×10⁻⁶ and about 1×10⁻¹², between about1×10⁻⁶ and about 1×10⁻¹¹, between about 1×10⁻⁶ and about 1×10⁻¹⁰,between about 1×10⁻⁶ and about 1×10⁻⁹, between about 1×10⁻⁶ and about1×10⁻⁸, between 1×10⁻⁶ and about 1×10⁻⁷, between about 1×10⁻⁷ and about1×10⁻¹², between about 1×10⁻⁷ and about 1×10⁻¹¹, between about 1×10⁻⁷and about 1×10⁻¹⁰, between about 1×10⁻⁷ and about 1×10⁻⁹, between about1×10⁻⁷ and about 1×10⁻⁸, between about 1×10⁻⁸ and about 1×10⁻¹², betweenabout 1×10⁻⁸ and about 1×10⁻¹¹, between about 1×10⁻⁸ and about 1×10⁻¹⁰,or between about 1×10⁻⁸ and about 1×10⁻⁹. A reduced bioburdenchromatography resin produced by any of the methods described herein canhave a binding capacity that is at least 74% (e.g., at least 76%, atleast 78%, at least 80%, at least 82%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) or betweenabout 74% and 95%, between about 74% and about 95%, between about 76%and about 95%, at least about 78% and about 95%, between about 80% andabout 95%, or between about 74% and about 90%, between about 76% andabout 90%, between about 78% and about 90%, or between about 80% andabout 90% of the binding capacity of the same chromatography resin thathas not been treated to reduce its bioburden (e.g., has not beengamma-irradiated), when the same protein is used to test the bindingcapacity of both the chromatography resin produced by the methodsdescribed herein and the control, untreated chromatography resin.

Methods of Making a Reduced Bioburden Packed Chromatography Column

Also provided herein are methods of making a reduced packedchromatography column that includes providing a reduced bioburdenchromatography resin produced by any of the methods described herein andpacking the chromatography resin into a reduced bioburden column in anaseptic or reduced bioburden environment. In some embodiments, a reducedbioburden packed chromatography column can be produced by exposing acolumn including a packed chromatography resin and a liquid including atleast one alcohol (e.g., any of the exemplary liquids described herein,e.g., optionally further including at least one antioxidant and/orchelator) to a dose of gamma-irradiation sufficient to reduce thebioburden of the column and the packed chromatography resin, where theat least one alcohol is present in an amount sufficient to amelioratethe loss of binding capacity of the packed chromatography resin afterexposure to the dose of gamma-irradiation.

Also provided are reduced bioburden packed chromatography column(s)produced by the methods described herein. Any of the reduced bioburdenpacked chromatography column(s) produced by the methods described hereincan have a sterility assurance level of about or less than 1×10⁻⁶, aboutor less than 1×10⁻⁷, about or less than 10×10⁻⁸, about or less than1×10⁻¹¹, or about or less than 1×10⁻¹², or between about 1×10⁻⁶ andabout 1×10⁻¹², between about 1×10⁻⁶ and about 1×10⁻¹¹, between about1×10⁻⁶ and about 1×10⁻¹⁰, between about 1×10⁻⁶ and about 1×10⁻⁹, betweenabout 1×10⁻⁶ and about 1×10⁻⁸, between 1×10⁻⁶ and about 1×10⁻⁷, betweenabout 1×10⁻⁷ and about 1×10⁻¹², between about 1×10⁻⁷ and about 1×10⁻¹¹,between about 1×10⁻⁷ and about 1×10⁻¹⁰, between about 1×10⁻⁷ and about1×10⁻⁹, between about 1×10⁻⁷ and about 1×10⁻⁸, between about 1×10⁻⁸ andabout 1×10⁻¹², between about 1×10⁻⁸ and about 1×10⁻¹¹, between about1×10⁻⁸ and about 1×10⁻¹⁰, or between about 1×10⁻⁸ and about 1×10⁻⁹. Anyof the reduced bioburden packed chromatography column(s) produced by themethods described herein can contain at least one chromatography resinselected from the group of: anionic exchange chromatography resin,cationic exchange chromatography resin, affinity chromatography resin(e.g., any of the affinity chromatography resins described herein orknown in the art), hydrophilic interaction chromatography resin, andsize exclusion chromatography resin. For example, any of the reducedbioburden packed chromatography columns described herein can include anaffinity chromatography resin including a protein ligand (e.g., proteinA). A reduced bioburden packed chromatography column described hereincan include an anionic exchange chromatography resin (e.g., an anionicexchange chromatography resin including N-benzyl-N-methyl-ethanolaminegroups).

Methods of Performing Reduced Bioburden Chromatography

The methods described herein include the use of a reduced bioburdenpacked chromatography column provided herein and the processes describedherein include the use of one or two MCCSs that include at least onereduced bioburden packed chromatography column provided herein. Thegamma-irradiated chromatography resin can be any type of resin describedherein (or any type of chromatography resin known in the art).

The reduced bioburden packed chromatography column can be prepared usingany of the methods described herein. For example, the reduced bioburdenpacked chromatography column can be produced by packing a chromatographycolumn with a composition including a chromatography resin(s) and aliquid including at least one alcohol (e.g., any of such compositionsdescribed herein), and exposing the packed column to gamma-irradiation(e.g., using any of the exposures and conditions described herein). Inother examples, the reduced bioburden packed chromatography column canbe produced by exposing a container including a chromatography resin anda liquid including at least one alcohol (e.g., any of the exemplaryliquids described herein, e.g., optionally further including at leastone antioxidant agent and/or at least one chelator) to a dose ofgamma-irradiation and packing a chromatography column with resultingreduced bioburden chromatography resin. In such methods, thechromatography resin (present in the container during exposure togamma-irradiation) can be present as a slurry in the container, and thechromatography column is packed in a reduced bioburden hood. In othermethods, the chromatography resin present in the container with theliquid including at least alcohol (e.g., any of the liquids describedherein, e.g., optionally further including at least one antioxidantagent and/or at least one chelator) can be exposed to gamma-irradiationas a wetted or moist solid mixture in the container, and a slurry of theresulting reduced bioburden chromatography resin can be prepared using areduced bioburden buffer (e.g., prepared in a reduced bioburden hood),and the resulting slurry used to pack a chromatography column in areduced bioburden hood. In some of these examples, the chromatographycolumn, prior to packing, can be treated to reduce the bioburden (e.g.,autoclaved, gamma-irradiated, or exposure to ethylene oxide).

The reduced bioburden packed chromatography column used in any of themethods described herein can have a sterility assurance level (SAL) ofbetween about 1×10⁻³ and about 1×10⁻¹², between about 1×10⁻⁴ and about1×10⁻¹², between 1×10⁻⁵ and about 1×10⁻¹¹, between about 1×10⁻⁵ andabout 1×10⁻¹⁰, between about 1×10⁻⁵ and about 1×10⁻⁹, between about1×10⁻⁶ and about 1×10⁻⁹, or between about 1×10⁻⁶ and about 1×10⁻⁸,inclusive.

Reduced Bioburden Buffers

The methods and processes described herein can be performed using one ormore reduced bioburden buffers. As can be appreciated in the art, areduced bioburden buffer can be any type of buffer used in a cycle ofchromatography (e.g., a buffer used in any of the steps in a cycle ofchromatography or in any of the unit operations described herein).Exemplary methods for reducing the bioburden of a buffer includefiltration (0.2 μm-pore size filtration), autoclaving, andgamma-irradiation. Additional methods for reducing the bioburden of abuffer are known in the art. A reduced bioburden buffer can have asterility assurance level of between about 1×10⁻³ and about 1×10⁻¹²,between about 1×10⁻⁴ and about 1×10⁻¹², between 1×10⁻⁵ and about1×10⁻¹¹, between about 1×10⁻⁵ and about 1×10⁻¹⁰, between about 1×10⁻⁵and about 1×10⁻⁹, between about 1×10⁻⁶ and about 1×10⁻⁹, or betweenabout 1×10⁻⁶ and about 1×10⁻⁸, inclusive.

Recombinant Therapeutic Proteins

A recombinant protein as described herein can be a recombinanttherapeutic protein. Non-limiting examples of recombinant therapeuticproteins that can be produced by the methods provided herein includeimmunoglobulins (including light and heavy chain immunoglobulins,antibodies, or antibody fragments (e.g., any of the antibody fragmentsdescribed herein), enzymes (e.g., a galactosidase (e.g., analpha-galactosidase), Myozyme®, or Cerezyme®), proteins (e.g., humanerythropoietin, tumor necrosis factor (TNF), or an interferon alpha orbeta), or immunogenic or antigenic proteins or protein fragments (e.g.,proteins for use in a vaccine). The recombinant therapeutic protein canbe an engineered antigen-binding polypeptide that includes at least onemultifunctional recombinant protein scaffold (see, e.g., the recombinantantigen-binding proteins described in Gebauer et al., Current Opin.Chem. Biol. 13:245-255, 2009; and U.S. Patent Application PublicationNo. 2012/0164066 (herein incorporated by reference in its entirety)).Non-limiting examples of recombinant therapeutic proteins that areantibodies include: panitumumab, omalizumab, abagovomab, abciximab,actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab,alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab,apolizumab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab,bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab,eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab,etaracizumab, golimumab, infliximab, natalizumab, palivizumab,panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, andtrastuzumab. Additional examples of recombinant therapeutic antibodiesthat can be produced by the methods described herein are known in theart. Additional non-limiting examples of recombinant therapeuticproteins that can be produced/purified by the present methods include:alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa,antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetinalfa, tenecteplase, etanercept, coagulation factor IX, folliclestimulating hormone, interferon beta-1a, imiglucerase, dornase alfa,epoetin alfa, and alteplase.

A secreted, soluble recombinant therapeutic protein can be recoveredfrom the liquid culture medium (e.g., a first and/or second liquidculture medium) by removing or otherwise physically separating theliquid culture medium from the cells (e.g., mammalian cells). A varietyof different methods for removing liquid culture medium from cells(e.g., mammalian cells) are known in the art, including, for example,centrifugation, filtration, pipetting, and/or aspiration. The secretedrecombinant therapeutic protein can then be recovered and furtherpurified from the liquid culture medium using a variety of biochemicaltechniques including various types of chromatography (e.g., affinitychromatography, molecular sieve chromatography, cation exchangechromatography, anion exchange chromatography, or hydrophobicinteraction chromatography, or any combination thereof) and/orfiltration (e.g., molecular weight cut-off filtration).

Cycle of Chromatography

As is well-known in the art, the steps in a cycle of chromatography candiffer depending on the chromatography resin, the buffers used toperform each step in the cycle, and the biophysical characteristics ofthe target recombinant protein (e.g., recombinant therapeutic protein).For example, an affinity chromatography column can include the steps ofloading an affinity chromatography column with a fluid including thetarget recombinant protein, washing the column to remove unwantedbiological material (e.g., contaminating proteins and/or smallmolecules), eluting the target recombinant protein bound to the column,and re-equilibrating the column. A cycle of chromatography using acationic and/or anionic exchange chromatography column, where the targetrecombinant protein binds to the chromatography resin in the loadingstep, can include the steps of loading the column with a fluid includingthe target protein, washing the column to remove unwanted biologicalmaterial, eluting the target recombinant protein bound to the column,and re-equilibrating the column. In other examples, a cycle ofchromatography using a cationic and/or anionic exchange chromatographycolumn, where unwanted biological material binds to the chromatographyresin during the loading step, while the target recombinant protein doesnot, can include the steps of loading the column with a fluid includingthe target protein, collecting the target recombinant protein in theflow-through, and reequilibrating the column. As is well-known in theart, any of the single steps in a chromatography cycle can include asingle buffer or multiple buffers (e.g., two or more buffers), and oneor more of any of the single steps in a chromatography cycle can includea buffer gradient. Any of the combination of various well-known aspectsof a single cycle of chromatography can be used in these methods in anycombination, e.g., different chromatography resin(s), flow-rate(s),buffer(s), void volume(s) of the column, bed volume(s) of the column,volume(s) of buffer used in each step, volume(s) of the fluid includingthe target protein, and the number and types of buffer(s) used in eachstep.

Methods of Performing Reduced Bioburden Column Chromatography

Provided herein are methods of performing reduced bioburdenchromatography. These methods include providing a reduced bioburdenpacked chromatography column produced using any of the methods describedherein, and performing a column chromatography using the reducedbioburden packed chromatography column. The reduced bioburden packedchromatography column can include at least one of any of thechromatography resins described herein, in any combination. For example,the chromatography resin present in the reduced bioburden packedchromatography column can be an affinity resin including a proteinligand (e.g., protein A) or can include an anionic exchangechromatography resin. The reduced bioburden packed chromatography columncan have any of the exemplary internal volumes described herein. Thereduced bioburden packed chromatography column can have any shape (e.g.,a cylinder, near cylindrical shape, or ellipsoidal shape) describedherein or known in the art. The column chromatography performed in thesemethods can be used to purify or isolate a recombinant protein (e.g.,any of the recombinant therapeutic proteins described herein or known inthe art). In some examples, the reduced bioburden packed chromatographycolumn is part of a multi-column chromatography system (MCCS), e.g., canbe part of a periodic counter current chromatography system (PCCS).

The column chromatography performed can include at least one cycle ofchromatography described herein or known in the art. For example, the atleast one cycle of chromatography can include the steps of: capturingthe recombinant protein by exposing the chromatography resin with aliquid including a recombinant protein; washing the chromatography resinby exposing the chromatography resin with a wash buffer, eluting therecombinant protein by exposing the chromatography resin with an elutionbuffer; and regenerating the chromatography resin by exposing thechromatography resin to a regeneration buffer. In some examples, theliquid including the recombinant protein is a liquid culture medium(e.g., a liquid culture medium collected from a perfusion or batchculture) or a diluted liquid culture medium (e.g., a culture mediumdiluted in buffer).

The column chromatography can be performed using a closed and integratedsystem (e.g., any of the exemplary closed and integrated systemsdescribed herein or known in the art). For example, the columnchromatography can be performed using a closed and integrated system,where the buffer is reduced bioburden buffer. As is well-known in theart, reduced bioburden buffer can be produced using a variety ofdifferent methods (e.g., prepared by filtration, by autoclaving, or heattreatment).

The column chromatography can include two or more (e.g., 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more,25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more,55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more,85 or more, 90 or more, 95 or more, or 100 or more) cycles ofchromatography. In some examples, the column chromatography is performedcontinuously over a period of at least 3 days (e.g., at least 4 days, atleast 5 days, at least 6 days, at least 7 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 16 days, atleast 17 days, at least 18 days, at least 19 days, at least 20 days, atleast 21 days, at least 22 days, at least 23 days, at least 24 days, atleast 25 days, at least 30 days, at least 35 days, at least 40 days, atleast 45 days, at least 50 days, at least 55 days, at least 60 days, atleast 65 days, at least 70 days, at least 75 days, at least 80 days, atleast 85 days, at least 90 days, at least 95 days, or at least 100days).

In some embodiments, the chromatography resin in the reduced bioburdenpacked chromatography column has a percentage binding capacity ofbetween about 75% to about 100% (e.g., between about 76% and about 98%,between about 76% and about 96%, between about 76% and about 94%,between about 76% and about 92%, between about 76% and about 90%,between about 78% and about 100%, between about 78% and about 98%,between about 78% and about 96%, between about 78% and about 94%,between about 78% and about 92%, between about 78% and about 90%,between about 80% and about 100%, between about 80% and about 98%,between about 80% and about 96%, between about 80% and about 94%,between about 80% and about 92%, between about 80% and about 90%,between about 82% and about 100%, between about 82% and about 98%,between about 82% and about 96%, between about 82% and about 94%,between about 82% and about 92%, between about 82% and about 90%,between about 84% and about 100%, between about 84% and about 98%,between about 84% and about 96%, between about 84% and about 94%,between about 84% and about 92%, between about 84% and about 90%,between about 86% and about 100%, between about 86% and about 98%,between about 86% and about 96%, between about 86% and about 94%,between about 86% and about 92%, between about 88% and about 100%,between about 88% and about 98%, between about 88% and about 96%,between about 88% and about 94%, between about 90% and about 100%,between about 90% and about 98%, between about 90% and about 96%,between about 92% and about 100%, or between about 92% and about 98%) ascompared to the same resin not treated with gamma-irradiation (when thesame protein is used to test the binding capacity of both resins) (e.g.,assessed immediately after exposure to gamma-irradiation).

Integrated, Closed or Substantially Closed, and Continuous Processes forManufacturing of a Recombinant Protein

Provided herein are integrated, closed or substantially closed, andcontinuous processes for manufacturing a purified recombinant protein(e.g., a recombinant therapeutic protein). These processes includeproviding a liquid culture medium including a recombinant protein (e.g.,a recombinant therapeutic protein) that is substantially free of cells.

Some processes include continuously feeding the liquid culture mediuminto a multi-column chromatography system (MCCS) that includes at leastone reduced bioburden packed chromatography column provided herein,where these processes utilize reduced bioburden buffer, are integrated,and run continuously from the liquid culture medium to an eluate fromthe MCCS that is the purified recombinant protein (e.g., a therapeuticprotein drug substance).

Some processes include continuously feeding the liquid culture mediuminto a first MCCS (MCCS1), capturing the recombinant protein from theliquid culture medium using the MCCS1, producing an eluate from theMCCS1 that includes the recombinant protein and continuously feeding theeluate into a second MCCS (MCCS2), and continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, where at least one column in the MCCS1 and/or theMCCS2 is a reduced bioburden packed chromatography column providedherein, the processes utilize reduced bioburden buffer, are integrated,and run continuously from the liquid culture medium to the purifiedrecombinant protein.

In some examples, each of the chromatography columns used in the MCCS,MCCS1, and/or MCCS2 is a reduced bioburden packed chromatography columnprovided herein. Some embodiments further include a step of formulatingthe purified recombinant protein into a pharmaceutical composition.

The processes described herein provide continuous and time-efficientproduction of a purified recombinant protein from a liquid culturemedium including the recombinant protein. For example, the elapsed timebetween feeding a liquid culture medium including a therapeutic proteininto the MCCS or MCCS1 and eluting the recombinant protein from the MCCSor MCCS2, respectively, can be, e.g., between about 4 hours and about 48hours, inclusive, e.g., between about 4 hours and about 40 hours,between about 4 hours and about 35 hours, between about 4 hours andabout 30 hours, between about 4 hours and about 28 hours, between about4 hours and about 26 hours, between about 4 hours and about 24 hours,between about 4 hours and about 22 hours, between about 4 hours andabout 20 hours, between about 4 hours and about 18 hours, between about4 hours and about 16 hours, between about 4 hours and about 14 hours,between about 4 hours and about 12 hours, between about 6 hours andabout 12 hours, between about 8 hours and about 12 hours, between about6 hours and about 20 hours, between about 6 hours and about 18 hours,between about 6 hours and about 14 hours, between about 8 hours andabout 16 hours, between about 8 hours and about 14 hours, between about8 hours and about 12 hours, between about 10 hours and 20 hours, betweenabout 10 hours and 18 hours, between about 10 hours and 16 hours,between about 10 hours and 14 hours, between about 12 hours and about 14hours, between about 10 hours and about 40 hours, between about 10 hoursand about 35 hours, between about 10 hours and about 30 hours, betweenabout 10 hours and about 25 hours, between about 15 hours and about 40hours, between about 15 hours and about 35 hours, between about 15 hoursand about 30 hours, between about 20 hours and about 40 hours, betweenabout 20 hours and about 35 hours, or between about 20 hours and about30 hours, inclusive. In other examples, the elapsed time between feedingthe liquid culture medium including the recombinant protein into theMCCS or MCCS1 and eluting the recombinant protein from the MCCS orMCCS2, respectively, is, e.g., greater than about 4 hours and less thanabout 40 hours, inclusive, e.g., greater than about 4 hours and lessthan about 39 hours, about 38 hours, about 37 hours, about 36 hours,about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours,about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours,about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5hours, inclusive.

Non-limiting aspects of the MCCSs that can be used in any of theseprocesses (MCCS, MCCS1, and/or MCCS2) are described in U.S. ProvisionalPatent Application Ser. Nos. 61/775,060 and 61/856,390 (eachincorporated herein by reference).

Some exemplary processes do not utilize a holding step (e.g., do not usea reservoir (e.g., break tank) in the entire process). Others may use amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process. Any of the processes described herein can utilize amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process, where each break tank only holds a recombinant proteinfor a total time period of, e.g., between about 5 minutes and less thanabout 6 hours, inclusive, e.g., between about 5 minutes and about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive.

Some processes utilize one, two, three, four, five, or six reservoir(s)(e.g., break tank(s)) and can have a capacity that is, e.g., between 1mL and about 300 mL, inclusive, e.g., between 1 mL and about 280 mL,about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL,about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL,about 60 mL, about 40 mL, about 20 mL, or about 10 mL (inclusive). Anyreservoir(s) (e.g., break tank(s)) used (in any of the processesdescribed herein) to hold fluid before it is fed into the MCCS or MCCS1can have a capacity that is, e.g., between 1 mL and about 100%,inclusive, e.g., between 1 mL and about 90%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%,inclusive, of the loading volume of the first column of the MCCS orMCCS1. A reservoir(s) (e.g., break tanks(s)) can be used to hold eluatefrom MCCS1 before it enters into the MCCS2 and can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS2.

Various additional aspects of these processes are described in detailbelow and can be used in any combination in the processes providedherein without limitation. Exemplary aspects of the provided processesare described below; however, one skilled in the art will appreciatethat additional steps can be added to the processes described herein andother materials can be used to perform any of the steps of the processesdescribed herein.

Liquid Culture Medium

Liquid culture medium that includes a recombinant protein (e.g.,recombinant therapeutic protein) that is substantially free of cells canbe derived from any source. For example, the liquid culture medium canbe obtained from a recombinant cell culture (e.g., a recombinantbacterial, yeast, or mammalian cell culture). The liquid culture mediumcan be obtained from a fed-batch cell (e.g., mammalian cell) culture(e.g., a fed-batch bioreactor including a culture of mammalian cellsthat secrete the recombinant protein) or a perfusion cell (e.g.,mammalian cell) culture (e.g., a perfusion bioreactor including aculture of mammalian cells that secrete the recombinant protein). Theliquid culture medium can also be a clarified liquid culture medium froma culture of bacterial or yeast cells that secrete the recombinantprotein.

Liquid culture medium obtained from a recombinant cell culture can befiltered or clarified to obtain a liquid culture medium that issubstantially free of cells and/or viruses. Methods for filtering orclarifying a liquid culture medium in order to remove cells are known inthe art (e.g., 0.2-μm filtration and filtration using an AlternatingTangential Flow (ATF™) system). Recombinant cells can also be removedfrom liquid culture medium using centrifugation and removing thesupernatant that is liquid culture medium that is substantially free ofcells, or by allowing the cells to settle to the gravitational bottom ofa container (e.g., bioreactor) including the liquid culture medium, andremoving the liquid culture medium (the liquid culture medium that issubstantially free of cells) that is distant from the settledrecombinant cells.

The liquid culture medium can be obtained from a culture of recombinantcells (e.g., recombinant bacteria, yeast, or mammalian cells) producingany of the recombinant proteins (e.g., recombinant therapeutic proteins)described herein or known in the art. Some examples of any of theprocesses described herein can further include a step of culturingrecombinant cells (e.g., recombinant bacteria, yeast, or mammaliancells) that produce the recombinant protein (e.g., recombinanttherapeutic protein).

The liquid culture medium can be any of the types of liquid culturemedium described herein or known in the art. For example, the liquidculture medium can be selected from the group of: animal-derivedcomponent-free liquid culture medium, serum-free liquid culture medium,serum-containing liquid culture medium, chemically-defined liquidculture medium, and protein-free liquid culture medium. In any of theprocesses described herein, a liquid culture medium obtained from aculture can be diluted by addition of a second fluid (e.g., a buffer)before it is fed into the MCCS or MCCS1.

The liquid culture medium including a recombinant protein that issubstantially free of cells can be stored (e.g., at a temperature belowabout 15° C. (e.g., below about 10° C., below about 4° C., below about0° C., below about −20° C., below about −50° C., below about −70° C., orbelow about −80° C.) for at least 1 day (e.g., at least about 2 days, atleast about 5 days, at least about 10 days, at least about 15 days, atleast about 20 days, or at least about 30 days) prior to feeding theliquid culture medium into the MCCS or MCCS1. Alternatively, in someexamples the liquid culture medium is fed into the MCCS or MCCS1directly from a bioreactor (e.g., fed into the MCCS or MCCS1 directlyfrom the bioreactor after a filtering or clarification step).

Multi-Column Chromatography Systems

The processes described herein include the use of a MCCS or two or more(e.g., two, three, four, five, or six) multi-column chromatographysystems (MCCSs) (e.g., an MCCS1 and MCCS2). A MCCS can include two ormore chromatography columns, two or more chromatographic membranes, or acombination of at least one chromatography column and at least onechromatographic membrane. In non-limiting examples, a MCCS (e.g., MCCS,MCCS1, and/or MCCS2 in any of the processes herein) can include fourchromatographic columns, three chromatographic columns and achromatographic membrane, three chromatographic columns, twochromatographic columns, two chromatographic membranes, and twochromatographic columns and one chromatographic membrane. Additionalexamples of combinations of chromatography columns and/orchromatographic membranes can be envisioned for use in an MCCS (e.g.,MCCS, MCCS1, and/or MCCS2 in any of the processes described herein) byone skilled in the art without limitation. The individual chromatographycolumns and/or chromatographic membranes present in a MCCS can beidentical (e.g., have the same shape, volume, resin, capture mechanism,and unit operation), or can be different (e.g., have one or more of adifferent shape, volume, resin, capture mechanism, and/or unitoperation). The individual chromatography column(s) and/orchromatographic membrane(s) present in a MCCS (e.g., MCCS, MCCS1, and/orMCCS2 in any of the processes described herein) can perform the sameunit operation (e.g., the unit operation of capturing, purifying, orpolishing) or different unit operations (e.g., different unit operationsselected from, e.g., the group of capturing, purifying, polishing,inactivating viruses, adjusting the ionic concentration and/or pH of afluid including the recombinant protein, and filtering). For example, inexamples of the processes described herein, at least one chromatographycolumn and/or chromatographic membrane in the MCCS or MCCS1 performs theunit operation of capturing the recombinant protein.

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a resin volumeof, e.g., between about 1 mL and about 2 mL, about 5 mL, about 10 mL,about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL,about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about95 mL, or about 100 mL, inclusive. The one or more chromatographycolumn(s) that can be present in an MCCS (e.g., present in the MCCS,MCCS1, and/or MCCS2) can have a resin volume of between about 2 mL toabout 100 mL, between about 2 mL and about 90 mL, between about 2 mL andabout 80 mL, between about 2 mL and about 70 mL, between about 2 mL andabout 60 mL, between about 2 mL and about 50 mL, between about 5 mL andabout 50 mL, between about 2 mL and about 45 mL, between about 5 mL andabout 45 mL, between about 2 mL and about 40 mL, between about 5 mL andabout 40 mL, between about 2 mL and about 35 mL, between about 5 mL andabout 35 mL, between about 2 mL and about 30 mL, between about 5 mL andabout 30 mL, between about 2 mL and about 25 mL, between about 5 mL andabout 25 mL, between about 15 mL and about 60 mL, between about 10 mLand about 60 mL, between about 10 mL and about 50 mL, and between about15 mL and about 50 mL. The one or more chromatography column(s) in anMCCS (e.g., the MCCS, MCCS1, and/or MCCS2) used in any of the processesdescribed herein can have the substantially the same resin volume or canhave different resin volumes. The flow rate used for the one or morechromatography column(s) in an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) can be, e.g., between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5 mLminute and about 14 mL/minute, between about 1.0 mL/minute and about25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute).

The one or more chromatography column (s) in an MCCS (e.g., MCCS, MCCS1,and/or MCCS2) can have substantially the same shape or can havesubstantially different shapes. For example, the one or morechromatography column(s) in an MCCS (e.g., in the MCCS, MCCS1, and/orMCCS2) can have substantially the shape of a circular cylinder or canhave substantially the same shape of an oval cylinder.

The one or more chromatographic membrane(s) that can be present in anMCCS (e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a bedvolume of, e.g., between about 1 mL to about 500 mL (e.g., between about1 mL to about 475 mL, between about 1 mL to about 450 mL, between about1 mL to about 425 mL, between about 1 mL to about 400 mL, between about1 mL to about 375 mL, between about 1 mL to about 350 mL, between about1 mL to about 325 mL, between about 1 mL to about 300 mL, between about1 mL to about 275 mL, between about 1 mL to about 250 mL, between about1 mL to about 225 mL, between about 1 mL to about 200 mL, between about1 mL to about 175 mL, between about 1 mL to about 150 mL, between about1 mL to about 125 mL, between about 1 mL to about 100 mL, between about2 mL to about 100 mL, between about 5 mL to about 100 mL, between about1 mL to about 80 mL, between about 2 mL to about 80 mL, between about 5mL to about 80 mL, between about 1 mL to about 60 mL, between about 2 mLto about 60 mL, between about 5 mL to about 60 mL, between about 1 mL toabout 40 mL, between about 2 mL to about 40 mL, between about 5 mL toabout 40 mL, between about 1 mL to about 30 mL, between about 2 mL toabout 30 mL, between about 5 mL to about 30 mL, between about 1 mL andabout 25 mL, between about 2 mL and about 25 mL, between about 1 mL andabout 20 mL, between about 2 mL and about 20 mL, between about 1 mL andabout 15 mL, between about 2 mL and about 15 mL, between about 1 mL andabout 10 mL, or between about 2 mL and about 10 mL).

One or more (e.g., three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, ortwenty-four) different types of reduced bioburden buffer can be employedduring the use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein. As is known in the art, the one or more types ofreduced bioburden buffer used in the MCCS, MCCS1, and/or MCCS2 in theprocesses described herein will depend on the resin present in thechromatography column(s) and/or the chromatographic membrane(s) of theMCCS, MCCS1, and/or MCCS2, the biophysical properties of the recombinantprotein, and unit operation (e.g., any of the exemplary unit operationsdescribed herein) performed by the specific chromatography column(s)and/or chromatography membranes of the MCCS, MCCS1, and/or MCCS2. Thevolume and type of buffer employed during the use of the MCCS, MCCS1,and/or MCCS2 in any of the processes described herein can also bedetermined by one skilled in the art (e.g., discussed in more detailbelow). For example, the volume and type(s) of buffer employed duringthe use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein can be chosen in order to optimize one or more of thefollowing in the purified recombinant protein (e.g., recombinant proteindrug product): the overall yield of recombinant protein, the activity ofthe recombinant protein, the level of purity of the recombinant protein,and the removal of biological contaminants from a fluid including therecombinant protein (e.g., liquid culture medium) (e.g., absence ofactive viruses, mycobacteria, yeast, bacteria, or mammalian cells).

The MCCS, MCCS1, and/or MCCS2 can be a periodic counter currentchromatography system (PCCS). A PCCS can, e.g., include two or morechromatography columns (e.g., three columns or four columns) that areswitched in order to allow for the continuous elution of recombinantprotein from the two or more chromatography columns. A PCCS can includetwo or more chromatography columns, two or more chromatographicmembranes, or at least one chromatographic column and at least onechromatographic membrane. A column operation (cycle) generally consistsof the load, wash, eluate, and regeneration steps. In PCCSs, multiplecolumns are used to run the same steps discretely and continuously in acyclic fashion. Since the columns are operated in series, the flowthrough and wash from one column is captured by another column. Thisunique feature of PCCSs allows for loading of the resin close to itsstatic binding capacity instead of to the dynamic binding capacity, asis typical during batch mode chromatography. As a result of thecontinuous cycling and elution, fluid entering a PCCS is processedcontinuously, and the eluate including recombinant protein iscontinuously produced.

Column-switching strategy is employed to advance from one step toanother in a PCCS cycle. Examples of column switching that can be usedin a PCCS are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390. For example, a column switching method canemploy two automated switching operations per column: the first of whichis related to the initial product breakthrough, while the secondcoincides with column saturation. The determination of when the columnswitching operations should take place can be determined by monitoringthe recombinant protein concentration (e.g., monitoring performed by UVmonitoring) in the eluate from each chromatography column present in aPCCS. For example, column switching can be determined by any PAT toolcapable of in-line measurement of recombinant protein concentration withfeedback control. The PAT tool is capable of real-time in-linemeasurement of recombinant protein concentration with feedback control.As in known in the art, column switches can also be designed based ontime or the amount of fluid (e.g., buffer) passed through the one ormore chromatography column(s) and/or chromatographic membranes in theMCCS, MCCS1, and/or MCCS2.

In PCCSs, the residence time (RT) of the recombinant protein on the eachchromatography column and/or chromatographic membrane present in thePCCS can be decreased without increasing the column/membrane sizebecause the breakthrough from the first column/membrane can be capturedon another column/membrane in the PCCS. A continuous process system canbe designed to process liquid culture medium at any perfusion rate (D)by varying the column/membrane volume (V) and RT using the equation of:V=D*RT.

The one or more unit operations that can be performed by the MCCS or theMCC1 and/or MCCS2 used in the presently described processes include, forexample, capturing the recombinant protein, inactivating viruses presentin a fluid including the recombinant protein, purifying the recombinantprotein, polishing the recombinant protein, holding a fluid includingthe recombinant protein (e.g., using any of the exemplary break tank(s)described herein), filtering or removing particulate material and/orcells from a fluid including the recombinant protein, and adjusting theionic concentration and/or pH of a fluid including the recombinantprotein.

In some embodiments, the MCCS or the MCCS1 includes at least onechromatographic column and/or chromatographic membrane that performs theunit operation of capturing the recombinant protein. The unit operationof capturing can be performed using at least one chromatography columnand/or chromatography resin, e.g., that utilizes a capture mechanism.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Capturing can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to capture arecombinant protein are described herein. Additional examples of resinsthat can be used to capture a recombinant protein are known in the art.

The unit operation of inactivating viruses present in a fluid includingthe recombinant protein can be performed using a MCCS, MCCS1, and/orMCCS2 (e.g., that include(s), e.g., a chromatography column, achromatography membrane, or a holding tank that is capable of incubatinga fluid including the recombinant protein at a pH of between about 3.0to 5.0 (e.g., between about 3.5 to about 4.5, between about 3.5 to about4.25, between about 3.5 to about 4.0, between about 3.5 to about 3.8, orabout 3.75) for a period of at least 30 minutes (e.g., a period ofbetween about 30 minutes to 1.5 hours, a period of between about 30minutes to 1.25 hours, a period of between about 0.75 hours to 1.25hours, or a period of about 1 hour)).

The unit operation of purifying a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS2) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that utilizes a capture system.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Purifying can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to purify arecombinant protein are described herein. Additional examples of resinsthat can be used to purify a recombinant protein are known in the art.

The unit operation of polishing a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that can be used to perform cationexchange, anion exchange, molecular sieve chromatography, or hydrophobicinteraction chromatography. Non-limiting resins that can be used topolish a recombinant protein are described herein. Additional examplesof resins that can be used to polish a recombinant protein are known inthe art.

The unit operation of holding a fluid including the recombinant proteincan be performed using an MCCS (e.g., a MCCS, MCCS1, and/or MCCS2) thatincludes at least one reservoir (e.g., a break tank) or a maximum of 1,2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in the MCCS or theMCCS1 and MCCS2 combined. For example, the reservoir(s) (e.g., breaktank(s)) that can be used to achieve this unit operation can each have avolume of between about 1 mL to about 1 L (e.g., between about 1 mL toabout 800 mL, between about 1 mL to about 600 mL, between about 1 mL toabout 500 mL, between about 1 mL to about 400 mL, between about 1 mL toabout 350 mL, between about 1 mL to about 300 mL, between about 10 mLand about 250 mL, between about 10 mL and about 200 mL, between about 10mL and about 150 mL, or between about 10 mL to about 100 mL). Thereservoir(s) (e.g., break tank(s)) used in the processes describedherein can have a capacity that is, e.g., between 1 mL and about 300 mL,inclusive, e.g., between 1 mL and about 280 mL, about 260 mL, about 240mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL,about 20 mL, or about 10 mL, inclusive. Any of the reservoir(s) (e.g.,break tank(s)) used (in any of the processes described herein) to holdfluid before it enters into the MCCS or MCCS1 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, between about 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS or MCCS1. Any of the reservoir(s) (e.g.,break tanks(s)) used to hold a eluate from MCCS1 (including therecombinant protein) before it enters the MCCS2 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between about 1mL and about 90%, about 80%, about 70%, about 60%, about 50%, about 40%,about 30%, about 20%, about 10%, or about 5%, inclusive, of the loadingvolume of the first column of the MCCS2.

The reservoir(s) (e.g., break tank(s)) can each hold the fluid includingthe recombinant protein for at least 10 minutes (e.g., at least 20minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 4 hours, or at least 6 hours). In other examples, the reservoir(s)(e.g., break tank(s)) only holds a recombinant protein for a total timeperiod of, e.g., between about 5 minutes and less than about 6 hours,inclusive, e.g., between about 5 minutes and about 5 hours, about 4hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes,inclusive. The reservoir(s) (e.g., break tank(s)) can be used to bothhold and refrigerate (e.g., at a temperature of less than 25° C., lessthan 15° C., or less than 10° C.) the fluid including the recombinantprotein. The reservoir can have any shape, including a circularcylinder, an oval cylinder, or an approximately rectangular sealed andnonpermeable bag.

The unit operations of filtering a fluid including the recombinantprotein can be performed using an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) that includes, e.g., a filter, or a chromatography column orchromatographic membrane that includes a molecular sieve resin. As isknown in the art, a wide variety of submicron filters (e.g., a filterwith a pore size of less than 1 μm, less than 0.5 μm, less than 0.3 μm,about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80 nm, lessthan 60 nm, less than 40 nm, less than 20 nm, or less than 10 nm) areavailable in the art that are capable of removing any precipitatedmaterial and/or cells (e.g., precipitated, unfolded protein;precipitated, unwanted host cell proteins; precipitated lipids;bacteria; yeast cells; fungal cells; mycobacteria; and/or mammaliancells). Filters having a pore size of about 0.2 μm or less than 0.2 μmare known to effectively remove bacteria from the fluid including therecombinant protein. As is known in the art, a chromatography column ora chromatographic membrane including a molecular sieve resin can also beused in an MCCS (e.g., the MCCS, MCCS1, and/or MCCS2) to perform theunit operation of filtering a fluid including a recombinant protein.

The unit operations of adjusting the ionic concentration and/or pH of afluid including the recombinant protein can be performed using a MCCS(e.g., a MCCS, a MCCS1, and/or a MCCS2) that includes and utilizes abuffer adjustment reservoir (e.g., an in-line buffer adjustmentreservoir) that adds a new buffer solution into a fluid that includesthe recombinant protein (e.g., between columns within the MCCS, MCCS1,and/or MCCS2, or after the last column in a penultimate MCCS (e.g., theMCCS1) and before the fluid including the recombinant protein is fedinto the first column of the next MCCS (e.g., the MCCS2)). As can beappreciated in the art, the in-line buffer adjustment reservoir can beany size (e.g., greater than 100 mL) and can include any bufferedsolution (e.g., a buffered solution that has one or more of: anincreased or decreased pH as compared to the fluid including therecombinant protein, an increased or decreased ionic (e.g., salt)concentration compared to the fluid including the recombinant protein,and/or an increased or decreased concentration of an agent that competeswith the recombinant protein for binding to resin present in at leastone chromatographic column or at least one chromatographic membrane inan MCCS (e.g., the MCCS, MCCS1, and/or MCCS2)).

The MCCS, MCCS1, and/or MCCS2 can perform two or more unit operations.For example, the MCCS, MCCS1, and/or MCCS2 can each perform at least thefollowing unit operations: capturing the recombinant protein andinactivating viruses present in the fluid including the recombinantprotein; capturing the recombinant protein, inactivating viruses presentin the fluid including the recombinant protein, and adjusting the ionicconcentration and/or pH of a liquid including the recombinant protein;purifying the recombinant protein and polishing the recombinant protein;purifying the recombinant protein, polishing the recombinant protein,and filtering a fluid including the recombinant protein or removingprecipitates and/or particular matter from a fluid including therecombinant protein; and purifying the recombinant protein, polishingthe recombinant protein, filtering a fluid including the recombinantprotein or removing precipitates and/or particulate matter from a fluidincluding the recombinant protein, and adjusting the ionic concentrationand/or pH of a liquid including the recombinant protein.

Capturing the Recombinant Protein

The present processes include a step of capturing the recombinantprotein using a MCCS or MCCS1. As can be appreciated in the art, theliquid culture medium including the recombinant protein can becontinuously fed onto the MCCS or MCCS1 using a variety of differentmeans. For example, the liquid culture medium can be actively pumpedinto the MCCS or MCCS1, or the liquid culture medium can be fed into theMCCS or MCCS1 using gravitational force. The liquid culture medium canbe stored in a reservoir (e.g., a holding tank) before it is fed intothe MCCS or MCCS1 or the liquid culture medium can be actively pumpedfrom a bioreactor including a culture of cells (e.g., mammalian cellsthat secrete the recombinant protein into the culture medium) into theMCCS or MCCS1.

The liquid culture medium can be fed (loaded) into the MCCS or MCCS1 ata flow rate of between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute). Theliquid culture medium including the recombinant protein can be derivedfrom any of the exemplary sources described herein or known in the art.

Some examples further include the optional step of filtering the liquidculture medium before it is fed into the MCCS or MCCS1. Any of theexemplary means of filtering a liquid culture medium or a fluidincluding the recombinant protein described herein, or any filtrationmeans known in the art, can be used to filter the liquid culture mediumincluding the recombinant protein before it is fed into the MCCS orMCCS1.

In the processes described herein, the capturing of the recombinantprotein from the liquid culture medium is performed using the MCCS orMCCS1. As can be appreciated in the art, in order to achieve the captureof the recombinant protein, at least one chromatographic column or atleast one chromatographic membrane in the MCCS or MCCS1 must include aresin that utilizes a capturing mechanism (e.g., any of the exemplarycapturing mechanisms described herein), or includes a resin capable ofperforming cation exchange, anion exchange, molecular sieve, orhydrophobic interaction chromatography. For example, if the recombinantprotein is an antibody or an antibody fragment, the capturing system canbe a protein A-binding capturing mechanism or an antigen-bindingcapturing mechanism (where the capturing antigen is specificallyrecognized by the recombinant antibody or antibody fragment). If therecombinant protein is an enzyme, the capturing mechanism can use anantibody or antibody fragment that specifically binds to the enzyme tocapture the recombinant enzyme, a substrate of the enzyme to capture therecombinant enzyme, a cofactor of the enzyme to capture the recombinantenzyme, or, if the recombinant enzyme includes a tag, a protein, metalchelate, or antibody (or antibody fragment) that specifically binds tothe tag present in the recombinant enzyme. Non-limiting resins that canbe used to capture a recombinant protein are described herein andadditional resins that can be used to capture a recombinant protein areknown in the art. One non-limiting example of resin that utilizes aprotein A-binding capture mechanism is Mab Select SuRe™ resin (GEHealthcare, Piscataway, N.J.), JSR LifeSciences Amsphere ProA JWT203(Sunnyvale, Calif.), and Kaneka KanCap A (Osaka, Japan).

Exemplary non-limiting sizes and shapes of the chromatography column(s)or chromatographic membrane(s) present in the MCCS or MCCS1 that can beused to capture the recombinant protein are described herein. The liquidculture medium fed (loaded) into the MCCS or MCCS1 can include, e.g.,between about 0.05 mg/mL to about 100 mg/mL recombinant protein (e.g.,between about 0.1 mg/mL to about 90 mg/mL, between about 0.1 mg/mL toabout 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL, between about0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL,between about 0.1 mg/mL to about 40 mg/mL, between about 0.1 mg/mL toabout 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL,between about 0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL toabout 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mL recombinantprotein). The mean time required for the recombinant protein to bind tothe resin used to perform the unit operation of capturing can be, e.g.,between about 5 seconds to about 10 minutes (e.g., between about 10seconds to about 8 minutes, between about 10 seconds to about 7 minutes,between about 10 seconds to about 6 minutes, between about 10 seconds toabout 5 minutes, between about 30 seconds to about 5 minutes, betweenabout 1 minute to about 5 minutes, between about 10 seconds to about 4minutes, between about 30 seconds to about 4 minutes, or between about 1minute to about 4 minutes).

As can be appreciated in the art, in order to capture the recombinantprotein using the chromatography column(s) or chromatographicmembrane(s) present in the MCCS or MCCS1, one must perform thesequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1. Any of the exemplary flow rates, buffervolumes, and/or lengths of time allotted for each sequentialchromatographic step described herein can be used in the one or more ofthese different sequential chromatographic steps (e.g., one or more ofthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1 that are used for capturing the recombinantprotein). Non-limiting flow rates, buffer volumes, and/or lengths oftime allotted for each sequential chromatographic step that can be usedfor capturing chromatographic column(s) and/or chromatographicmembrane(s) in the MCCS or MCCS1 (e.g., a PCCS or PCCS1) are providedbelow. In addition, exemplary buffers that can be used in the MCCSand/or MCCS1 are described below.

The MCCS or MCCS1 including at least one chromatographic column and/orchromatographic membrane including a resin that can perform the unitoperation of capturing (e.g., any of exemplary resins that can be usedfor capturing described herein) can be loaded with the liquid culturemedium including a recombinant protein using any of loading flow rates(fed rates) described above. In some examples, a single chromatographiccolumn or single chromatographic membrane including a resin that iscapable of performing the unit operation of capturing is loaded in,e.g., between about 10 minutes to about 90 minutes (e.g., between about15 minutes and about 90 minutes, between about 20 minutes and 80minutes, between about 30 minutes and 80 minutes, between about 40minutes and about 80 minutes, between about 50 minutes and about 80minutes, and between about 60 minutes and 80 minutes). In some examples,wherein the MCCS or MCCS1 includes at least two chromatographic columnsthat include a resin that is capable of performing the unit operation ofcapturing in series, the time required to load two of thechromatographic columns in series is, e.g., between about 50 minutes toabout 180 minutes (e.g., between about 60 minutes and about 180 minutes,between about 70 minutes and about 180 minutes, between about 80 minutesand about 180 minutes, between about 90 minutes and about 180 minutes,between about 100 minutes and about 180 minutes, between about 110minutes and 150 minutes, and between about 125 minutes and about 145minutes).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane in the MCCS or MCCS1that includes a resin that is capable of performing the unit operationof capturing, the at least one chromatographic column or chromatographicmembrane is washed with at least one washing buffer. As can beappreciated in the art, the at least one (e.g., two, three, or four)washing buffer is meant to elute all or most of proteins that are notthe recombinant protein from the at least one chromatography column orchromatographic membrane, while not disturbing the interaction of therecombinant protein with the resin.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15× CV (e.g., between about 1× CV to about 14× CV, about 1× CV toabout 13× CV, about 1× CV to about 12× CV, about 1× CV to about 11× CV,about 2× CV to about 11× CV, about 3× CV to about 11× CV, about 4× CV toabout 11× CV, about 5× CV to about 11× CV, or about 5× CV to about 10×CV). The total time of the washing can be, e.g., between about 2 minutesto about 3 hours (e.g., between about 2 minutes to about 2.5 hours,between about 2 minutes to about 2.0 hours, between about 5 minutes toabout 1.5 hours, between about 10 minutes to about 1.5 hours, betweenabout 10 minutes to about 1.25 hours, between about 20 minutes to about1.25 hours, or between about 30 minutes to about 1 hour).

Following the washing of the at least one chromatographic column orchromatographic membrane in the MCCS or MCCS1 that includes a resin thatis capable of performing the unit operation of capturing, therecombinant protein is eluted from the at least one chromatographiccolumn or chromatographic membrane by passing an elution buffer throughthe at least one chromatographic column or chromatographic membrane inthe MCCS or MCCS1 that includes a resin that is capable of performingthe unit operation of capturing. The elution buffer can be passedthrough the at least one chromatography column or chromatographicmembrane that includes a resin that is capable of performing the unitoperation of capturing at a flow rate of between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, or between about 1.0 mL/minute and about 15.0 mL/minute). Thevolume of elution buffer used to elute the recombinant protein from eachof the at least one chromatographic column or chromatographic membraneincluding a resin that is capable of performing the unit operation ofpurifying can be, e.g., between about 1× column volume (CV) to about 15×CV (e.g., between about 1× CV to about 14× CV, about 1× CV to about 13×CV, about 1× CV to about 12× CV, about 1× CV to about 11× CV, about 2×CV to about 11× CV, about 3× CV to about 11× CV, about 4× CV to about11× CV, about 5× CV to about 11× CV, or about 5× CV to about 10× CV).The total time of the eluting can be, e.g., between about 2 minutes toabout 3 hours (e.g., between about 2 minutes to about 2.5 hours, betweenabout 2 minutes to about 2.0 hours, between about 2 minutes to about 1.5hours, between about 2 minutes to about 1.5 hours, between about 2minutes to about 1.25 hours, between about 2 minutes to about 1.25hours, between about 2 minutes to about 1 hour, between about 2 minutesand about 40 minutes, between about 10 minutes and about 40 minutes, orbetween about 20 minutes and about 40 minutes). Non-limiting examples ofelution buffers that can be used in these methods will depend on thecapture mechanism and/or the recombinant protein. For example, anelution buffer can include a different concentration of salt (e.g.,increased salt concentration), a different pH (e.g., an increased ordecreased salt concentration), or a molecule that will compete with therecombinant protein for binding to the resin that is capable ofperforming the unit operation of capturing. Examples of such elutionbuffers for each exemplary capture mechanism described herein are wellknown in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane in the MCCS or theMCCS1 that includes a resin that is capable of performing the unitoperation of capturing, and before the next volume of liquid culturemedium can be loaded onto the at least one chromatographic column orchromatographic membrane, the at least one chromatography column orchromatographic membrane must be equilibrated using an regenerationbuffer. The regeneration buffer can be passed through the at least onechromatography column or chromatographic membrane that includes a resinthat is capable of performing the unit operation of capturing at a flowrate of, e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, or between about 1.0 mL/minute andabout 15.0 mL/minute). The volume of regeneration buffer used toequilibrate the at least one chromatography column or chromatographicmembrane that includes a resin that is capable of performing the unitoperation of capturing can be, e.g., between about 1× column volume (CV)to about 15× CV (e.g., between about 1× CV to about 14× CV, about 1× CVto about 13× CV, about 1× CV to about 12× CV, about 1× CV to about 11×CV, about 2× CV to about 11× CV, about 3× CV to about 11× CV, about 2×CV to about 5× CV, about 4× CV to about 11× CV, about 5× CV to about 11×CV, or about 5× CV to about 10× CV).

In some of the processes described herein, the MCCS or MCCS1 includes areservoir that holds a fluid including the recombinant protein at low pH(e.g., a pH below 4.6, below 4.4, below 4.2, below 4.0, below 3.8, below3.6, below 3.4, below 3.2, or below 3.0) for, e.g., about 1 minute to1.5 hours (e.g., about 1 hour), and inactivates the viruses present in afluid including the recombinant protein. An example of a reservoir thatcan be used to perform the unit operation of inactivating viruses is astir flask (e.g., 500-mL stir flask, e.g., a 500-mL stir flask with aprogrammed stir plate) that is capable of holding a fluid including arecombinant protein for, e.g., about 1 minute to 1.5 hours, e.g., beforethe fluid including the recombinant protein is fed into the MCCS2. Thereservoir that is used to perform the unit operation of inactivation ofviruses can be a 500-mL stir flask with a programmed stir plate (e.g., astir plate programmed to mix (e.g., periodically mix) the fluid withinthe reservoir, e.g., every 4 hours). Another example of a reservoir thatcan be used to perform the unit operation of inactivation of viruses isa plastic bag (e.g., 500-mL plastic bag) that is capable of holding afluid including a recombinant protein for, e.g., about 1 minute to 1.5hours, e.g., before the fluid including the recombinant protein is fedinto the MCCS2. In some examples, the fluid including the recombinantprotein can already have a low pH (e.g., a pH below 4.6, below 4.4,below 4.2, below 4.0, below 3.8, below 3.6, below 3.4, below 3.2, orbelow 3.0) when it is fed into the reservoir that is used to perform theunit operation of viral inactivation. As can be appreciated by thoseskilled in the art, a variety of other means can be used to perform theunit operation of inactivating viruses. For example, UV irradiation of afluid including the recombinant protein can also be used to perform theunit operation of inactivating viruses. Non-limiting examples ofreservoirs that can be used to perform the unit operation ofinactivation of viruses present in a fluid including the recombinantprotein are described herein.

The MCCS or MCCS1 can include a PCCS including four chromatographycolumns, where at least three of the four chromatography columns performthe unit operation of capturing the recombinant protein from the liquidculture medium (e.g., using an MCCS that includes any of the at leastone chromatography columns that include a resin that is capable ofperforming the unit operation of capturing (e.g., any of those describedherein)). In these examples, the fourth-column of the PCC can performthe unit operation of inactivating viruses in a fluid that includes therecombinant protein (e.g., any of the exemplary columns described hereinthat can be used to achieve viral inactivation of a fluid including therecombinant protein).

In some examples, a fluid including the recombinant protein iscontinuously eluted from the MCCS1 (e.g., PCCS1), and is continuouslyfed into the MCCS2 (e.g., PCCS2). The percent of the recombinant proteinrecovered in the eluate of the MCCS or MCCS1 (e.g., PCCS or PCCS1) canbe, e.g., at least 70%, at least 72%, at least 74%, at least 76%, atleast 78%, at least 80%, at least 82%, at least 84%, at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, or atleast 98%). The eluate from the MCCS1 (e.g., PCCS1) can be fed into theMCCS2 (e.g., PCCS2) using a variety of means known in the art (e.g.,tubing). The eluate of the MCCS1 (e.g., PCCS1) can be fed into the MCCS2(e.g., PCCS2) at a flow rate of, e.g., between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, betweenabout 15 mL/minute to about 25 mL/minute, or between about 1.0 mL/minuteand about 15.0 mL/minute).

Some processes described herein can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the MCCS1 (e.g.,PCCS1) before it is fed into the MCCS2 (e.g., PCCS2). As describedherein, the ionic concentration and/or pH of the eluate from the MCCS1(e.g., PCCS1) can be adjusted (before it is fed into the MCCS2) byadding a buffer to the eluate (e.g., through the use of an in-linebuffer adjustment reservoir). The buffer can be added to the eluate fromthe MCCS1 at a flow rate of, e.g., between about 0.1 mL/minute to about15 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, or between about0.5 mL/minute to about 5 mL/minute).

The processes described herein can further include a step of holding orstoring (and optionally also refrigerating) the eluate from the MCCS1prior to feeding the eluate from the MCCS1 into the MCCS2. As describedherein, this holding or storing step can be performed using any of thereservoirs (e.g., back-up tanks) described herein.

The processes described herein can also include a step of filtering theeluate from the MCCS1 before the eluate is fed into the MCCS2. Any ofthe exemplary filters or methods for filtration described herein can beused to filter the eluate from the MCCS1 before the eluate is fed intothe MCCS2.

Polishing and Purifying the Recombinant Protein

The MCCS, MCCS1, and/or MCCS2 can be used to perform the unit operationof purifying and polishing the recombinant protein. For example, theMCCS2 can be used to perform the operation of purifying and polishingthe recombinant protein and the eluate from the MCCS2 is a protein drugsubstance. The MCCS, MCCS1, and/or MCCS2 can include at least one (e.g.,two, three, or four) chromatography column or chromatographic membranethat can be used to perform the unit operation of purifying arecombinant protein, and at least one (e.g., two, three, or four)chromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant protein.

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of purifying the recombinantprotein can include a resin that utilizes a capture mechanism (e.g., anyof the capture mechanisms described herein or known in the art), or aresin that can be used to perform anion exchange, cation exchange,molecular sieve, or hydrophobic interaction chromatography. The at leastone chromatography column or chromatographic membrane that can be usedto perform the unit of operation of polishing the recombinant proteincan include a resin that can be used to perform anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatography(e.g., any of the exemplary resins for performing anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatographydescribed herein or known in the art).

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit ofoperation of purifying the recombinant protein, and/or the size andshape of the at least one chromatographic membrane that can be used toperform the unit of operation of polishing the recombinant protein canany of combination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein. Ascan be appreciated by one skilled in the art, the step of purifying orpolishing a recombinant protein can, e.g., include the steps of loading,washing, eluting, and equilibrating the at least one chromatographycolumn or chromatographic membrane used to perform the unit of operationof purifying or polishing the recombinant protein. Typically, theelution buffer coming out of a chromatography column or chromatographicmembrane used to perform the unit operation of purifying includes therecombinant protein. Typically, the loading and/or wash buffer comingout of a chromatography column or chromatographic membrane used toperform the unit operation of polishing includes the recombinantprotein.

For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform unit operation ofpurifying the recombinant protein can have a volume of, e.g., betweenabout 2.0 mL to about 200 mL (e.g., between about 2.0 mL to about 180mL, between about 2.0 mL to about 160 mL, between about 2.0 mL to about140 mL, between about 2.0 mL to about 120 mL, between about 2.0 mL toabout 100 mL, between about 2.0 mL to about 80 mL, between about 2.0 mLto about 60 mL, between about 2.0 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 2.0 mL to about 30 mL, between about5.0 mL to about 30 mL, or between about 2.0 mL to about 25 mL). The flowrate of the fluid including the recombinant protein as it is loaded ontothe at least one chromatography column or at least one chromatographicthat can be used to perform the unit operation of purifying therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 0.1 mL/minute to about 2mL/minute, or about 0.2 mL/minute to about 4 mL/minute). Theconcentration of the recombinant protein in the fluid loaded onto the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 0.05 mg/mL to about 100 mg/mL recombinantprotein (e.g., between about 0.1 mg/mL to about 90 mg/mL, between about0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL,between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about 0.1mg/mL to about 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mLrecombinant protein). The resin in the at least one chromatographycolumn or chromatographic membrane used to perform unit operation ofpurifying can be a resin that can be used to perform anion exchange orcation exchange chromatography. The resin in the at least onechromatography column or chromatographic membrane that is used toperform the unit operation of purifying can be a cationic exchange resin(e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, N.J.).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein, the atleast one chromatographic column or chromatographic membrane is washedwith at least one washing buffer. As can be appreciated in the art, theat least one (e.g., two, three, or four) washing buffer is meant toelute all proteins that are not the recombinant protein from the atleast one chromatography column or chromatographic membrane, while notdisturbing the interaction of the recombinant protein with the resin orotherwise eluting the recombinant protein.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, or between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15× CV (e.g., between about 1× CV to about 14× CV, about 1× CV toabout 13× CV, about 1× CV to about 12× CV, about 1× CV to about 11× CV,about 2× CV to about 11× CV, about 3× CV to about 11× CV, about 4× CV toabout 11× CV, about 2.5× CV to about 5.0× CV, about 5× CV to about 11×CV, or about 5× CV to about 10× CV). The total time of the washing canbe, e.g., between about 2 minutes to about 3 hours (e.g., between about2 minutes to about 2.5 hours, between about 2 minutes to about 2.0hours, between about 5 minutes to about 1.5 hours, between about 10minutes to about 1.5 hours, between about 10 minutes to about 1.25hours, between about 20 minutes to about 1.25 hours, between about 30minutes to about 1 hour, between about 2 minutes and 10 minutes, betweenabout 2 minutes and 15 minutes, or between about 2 minutes and 30minutes).

Following the washing of the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein, the recombinant protein is eluted from the atleast one chromatographic column or chromatographic membrane by passingan elution buffer through the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein. The elution buffer can be passed through the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteinat a flow rate of between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5mL/minute and about 6.0 mL/minute, between about 1.0 mL/minute and about5.0 mg/minute, between about 0.5 mL minute and about 14 mL/minute,between about 1.0 mL/minute and about 25.0 mL/minute, or between about1.0 mL/minute and about 15.0 mL/minute). The volume of elution bufferused to elute the recombinant protein from each of the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein can be,e.g., between about 1× column volume (CV) to about 25× CV (e.g., betweenabout 1× CV to about 20× CV, between about 15× CV and about 25× CV,between about 1× CV to about 14× CV, about 1× CV to about 13× CV, about1× CV to about 12× CV, about 1× CV to about 11× CV, about 2× CV to about11× CV, about 3× CV to about 11× CV, about 4× CV to about 11× CV, about5× CV to about 11× CV, or about 5× CV to about 10× CV). The total timeof the eluting can be, e.g., between about 2 minutes to about 3 hours(e.g., between about 2 minutes to about 2.5 hours, between about 2minutes to about 2.0 hours, between about 2 minutes to about 1.5 hours,between about 2 minutes to about 1.5 hours, between about 2 minutes toabout 1.25 hours, between about 2 minutes to about 1.25 hours, betweenabout 2 minutes to about 1 hour, between about 2 minutes and about 40minutes, between about 10 minutes and about 40 minutes, between about 20minutes and about 40 minutes, or between about 30 minutes and 1.0 hour).Non-limiting examples of elution buffers that can be used in thesemethods will depend on the resin and/or the biophysical properties ofthe recombinant protein. For example, an elution buffer can include adifferent concentration of salt (e.g., increased salt concentration), adifferent pH (e.g., an increased or decreased salt concentration), or amolecule that will compete with the recombinant protein for binding tothe resin. Examples of such elution buffers for each of the exemplarycapture mechanisms described herein are well known in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane used to perform theunit operation of purifying the recombinant protein, and before the nextvolume of fluid including a recombinant protein can be loaded onto theat least one chromatographic column or chromatographic membrane, the atleast one chromatography column or chromatographic membrane must beequilibrated using an regeneration buffer. The regeneration buffer canbe passed through the at least one chromatography column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein at a flow rate of, e.g., between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, orbetween about 1.0 mL/minute and about 15.0 mL/minute). The volume ofregeneration buffer used to equilibrate the at least one chromatographycolumn or chromatographic membrane that includes a resin that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 1× column volume (CV) to about 15× CV (e.g.,between about 1× CV to about 14× CV, between about 1× CV to about 13×CV, between about 1× CV to about 12× CV, between about 1× CV to about11× CV, between about 2× CV to about 11× CV, between about 3× CV toabout 11× CV, between about 2× CV to about 5× CV, between about 2.5× CVto about 7.5× CV, between about 4× CV to about 11× CV, between about 5×CV to about 11× CV, or between about 5× CV to about 10× CV). Theconcentration of recombinant protein in the eluate of the at least onechromatography column or chromatographic membrane used to perform theunit operation of purifying the recombinant protein can be, e.g.,between about 0.05 mg/mL to about 100 mg/mL recombinant protein (e.g.,between about 0.1 mg/mL to about 90 mg/mL, between about 0.1 mg/mL toabout 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL, between about0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL,between about 0.1 mg/mL to about 40 mg/mL, between about 2.5 mg/mL andabout 7.5 mg/mL, between about 0.1 mg/mL to about 30 mg/mL, betweenabout 0.1 mg/mL to about 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL,between about 0.1 mg/mL to about 15 mg/mL, between about 0.5 mg/mL toabout 15 mg/mL, between about 0.1 mg/mL to about 10 mg/mL, or betweenabout 0.5 mg/mL to about 10 mg/mL recombinant protein).

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of polishing the recombinantprotein can include a resin that can be used to perform cation exchange,anion exchange, or molecular sieve chromatography. As can be appreciatedin the art, polishing a recombinant protein using the at least onechromatography column or chromatography membrane that can be used toperform the unit operation of polishing the recombinant protein caninclude, e.g., the steps of loading, chasing, and regenerating the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing the recombinant protein.For example, when the steps of loading, chasing, and regenerating areused to perform the polishing, the recombinant protein does not bind theresin in the at least one chromatography column or chromatographymembrane that is used to perform the unit operation of polishing therecombinant protein, and the recombinant protein is eluted from the atleast one chromatography column or chromatographic membrane in theloading and chasing steps, and the regenerating step is used to removeany impurities from the at least one chromatography column orchromatographic membrane before additional fluid including therecombinant protein can be loaded onto the at least one chromatographycolumn or chromatographic membrane. Exemplary flow rates and buffervolumes to be used in each of the loading, chasing, and regeneratingsteps are described below.

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit operationof polishing the recombinant protein, and/or the size and shape of theat least one chromatographic membrane that can be used to perform theunit operation of polishing the recombinant protein can any ofcombination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein.For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein can have a volume of, e.g., betweenabout 0.5 mL to about 200 mL (e.g., between about 0.5 mL to about 180mL, between about 0.5 mL to about 160 mL, between about 0.5 mL to about140 mL, between about 0.5 mL to about 120 mL, between about 0.5 mL toabout 100 mL, between about 0.5 mL to about 80 mL, between about 0.5 mLto about 60 mL, between about 0.5 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 0.5 mL to about 30 mL, between about5.0 mL to about 30 mL, between about 0.5 mL to about 25 mL, betweenabout 0.2 mL to about 10 mL, or between about 0.2 mL to about 5 mL). Theflow rate of the fluid including the recombinant protein as it is loadedonto the at least one chromatography column or chromatographic membranethat can be used to perform the unit operation of polishing therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 2 mL/minute and about 6mL/minute, between about 0.1 mL/minute to about 2 mL/minute, or about0.2 mL/minute to about 4 mL/minute). The total volume of fluid includinga recombinant protein loaded onto the at least one chromatography columnor chromatographic membrane that can be used to perform the unitoperation of polishing the recombinant protein can be, e.g., betweenabout 1.0 mL to about 250 mL (e.g., between about 1.0 mL to about 225mL, between about 1.0 mL to about 200 mL, between about 1.0 mL to about175 mL, between about 1.0 mL to about 150 mL, between about 100 mL toabout 125 mL, between about 100 mL to about 150 mL, between about 1.0 mLto about 150 mL, between about 1.0 mL to about 125 mL, between about 1.0mL to about 100 mL, between about 1.0 mL to about 75 mL, between about1.0 mL to about 50 mL, or between about 1.0 mL to about 25 mL). Theresin in the at least one chromatography column or chromatographicmembrane used to perform the polishing can be an anion exchange orcation exchange resin. The resin in the at least one chromatographycolumn or chromatographic membrane that is used to perform the unitoperation of polishing can be a cationic exchange resin (e.g.,Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, a chasing step is performed (e.g., a chasebuffer is passed through the at least one chromatography membrane orchromatographic membrane to collect the recombinant protein which doesnot substantially bind to the at least one chromatography column orchromatographic membrane). In these examples, the chase buffer can bepassed through the at least one chromatography column or chromatographicmembrane at a flow rate of between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of chase buffer used can be, e.g., between about1× column volume (CV) to about 100× CV (e.g., between about 1× CV toabout 90× CV, between about 1× CV to about 80× CV, between about 1× CVto about 70× CV, between about 1× CV to about 60× CV, between about 1×to about 50× CV, between about 1× CV to about 40× CV, between about 1×CV to about 30× CV, between about 1× CV to about 20× CV, between about1× CV to about 15× CV, between about 5× CV to about 20× CV, betweenabout 5 × CV to about 30× CV, between about 1× CV to about 14× CV, about1× CV to about 13× CV, about 1× CV to about 12× CV, about 1× CV to about11× CV, about 2× CV to about 11× CV, about 3× CV to about 11× CV, about4× CV to about 11× CV, about 2.5× CV to about 5.0× CV, about 5× CV toabout 11× CV, or about 5× CV to about 10× CV). The total time of thechasing can be, e.g., between about 1 minute to about 3 hours (e.g.,between about 1 minute to about 2.5 hours, between about 1 minute toabout 2.0 hours, between about 1 minute to about 1.5 hours, betweenabout 2 minutes to about 1.5 hours, between about 1 minute to about 1.25hours, between about 2 minutes to about 1.25 hours, between about 1minute to about 5 minutes, between about 1 minute to about 10 minutes,between about 2 minutes to about 4 minutes, between about 30 minutes toabout 1 hour, between about 2 minutes to about 10 minutes, between about2 minutes to about 15 minutes, or between about 2 minutes to about 30minutes). The combined concentration of recombinant protein present inthe eluate coming through the column in the loading step and the chasingstep can be, e.g., between about 0.1 mg/mL to about 100 mg/mLrecombinant protein (e.g., between about 0.1 mg/mL to about 90 mg/mL,between about 0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL toabout 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL, between about0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL,between about 2.5 mg/mL and about 7.5 mg/mL, between about 0.1 mg/mL toabout 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL,between about 0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL toabout 10 mg/mL, between about 0.5 mg/mL to about 10 mg/mL, or betweenabout 1 mg/mL and about 5 mg/mL recombinant protein).

Following the chasing step and before the next volume of fluid includingthe recombinant protein can be loaded onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of polishing, the at least one chromatographycolumn or chromatographic membrane must be regenerated using aregeneration buffer. The regeneration buffer can be passed through theat least one chromatography column or chromatographic membrane that canbe used to perform the unit operation of polishing the recombinantprotein at a flow rate of, e.g., between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of regeneration buffer used to regenerate the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing can be, e.g., betweenabout 1× column volume (CV) to about 500× CV (e.g., between about 1× CVto about 450× CV, between about 1× CV to about 400× CV, between about 1×CV to about 350× CV, between about 1× CV to about 300× CV, between about1× CV to about 250× CV, between about 1× CV to about 200× CV, betweenabout 1× CV to about 150× CV, between about 1× CV to about 100 × CV,between about 1× CV to about 90× CV, between about 1× CV to about 80×CV, between about 1× CV to about 70× CV, between about 1× CV to about60× CV, between about 1× to about 50× CV, between about 1× CV to about40× CV, between about 1× CV to about 30× CV, between about 1× CV toabout 20× CV, between about 1× CV to about 15× CV, between about 5× CVto about 20× CV, between about 5× CV to about 30× CV, between about 1×CV to about 14× CV, about 1× CV to about 13× CV, about 1× CV to about12× CV, about 1× CV to about 11× CV, about 2× CV to about 11× CV, about3× CV to about 11× CV, about 4× CV to about 11× CV, about 2.5× CV toabout 5.0× CV, about 5× CV to about 11× CV, or about 5× CV to about 10×CV).

In other examples, the one or more chromatography column(s) and/orchromatographic membranes used to perform the unit operation ofpolishing include a resin that selectively binds or retains theimpurities present in a fluid including the recombinant protein, andinstead of regenerating the one or more column(s) and/or membrane(s),the one or more column(s) and/or membrane(s) are replaced (e.g.,replaced with a substantially similar column(s) and/or membrane(s)) oncethe binding capacity of the resin in the one or more column(s) and/ormembrane(s) has been reached or is substantially close to being reached.

In some examples of these processes described herein, the MCCS2 includesa PCCS including three chromatography columns and one chromatographicmembrane, e.g., where the three chromatography columns in the PCCSperform the unit operation of purifying the recombinant protein (e.g.,using at least one chromatography column(s) that can be used to performthe unit of operation of purifying the protein) and the chromatographicmembrane in the PCCS performs the unit operation of polishing therecombinant protein. In these examples, the chromatographic membrane inthe PCCS that can be used to perform the unit operation of polishing thetherapeutic protein can be any of the exemplary chromatographicmembranes described herein that can be used to perform the unitoperation of polishing the recombinant protein. Any of the columnswitching methods described herein can be used to determine when thefirst three chromatography columns and the chromatographic membrane inthe PCCS in this example can be switched.

Some embodiments of this example can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the threechromatographic columns in the PCCS before the eluate is fed into thechromatographic membrane in the PCCS. As described herein, the ionicconcentration and/or pH of the eluate from the three chromatographycolumns in PCCS can be adjusted (before it is fed into thechromatographic membrane in the PCCS in this example)) by adding abuffer to the eluate of the three chromatography columns in the PCCS(e.g., through the use of an in-line buffer adjustment reservoir). Thebuffer can be added to the eluate at a flow rate of, e.g., between about0.1 mL/minute to about 15 mL/minute (e.g., between about 0.1 mL/minuteto about 12.5 mL/minute, between about 0.1 mL/minute to about 10.0mL/minute, between about 0.1 mL/minute to about 8.0 mL/minute, betweenabout 0.1 mL/minute to about 6 mL/minute, between about 0.1 mL/minute to4 mL/minute, or between about 0.5 mL/minute to about 5 mL/minute).

These examples can further include a step of holding or storing theeluate from the three chromatography columns in the PCCS in this exampleprior to feeding the eluate into the chromatographic membrane(chromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). As described herein, this holdingor storing step can be performed using any of the reservoirs (e.g.,back-up tanks) described herein.

These examples can also include a step of filtering the eluate from thechromatographic membrane in the exemplary PCCS system (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). Any of the exemplary filters ormethods for filtration described herein can be used to filter the eluatefrom the chromatographic membrane in this exemplary PCCS (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein).

As can be appreciated by those in the art, the purified recombinantprotein can be periodically eluted from the MCCS or MCCS2 using any ofthe processes described herein. For example, any of the processesdescribed herein can elute the purified recombinant protein for aduration of, e.g., between about 30 seconds and about 5 hours (e.g.,between about 1 minute and about 4 hours, between about 1 minute andabout 3 hours, between about 1 minute and about 2 hours, between about 1minute or about 1.5 hours, between about 1 minute and about 1 hour, orbetween about 1 minute and about 30 minutes) at a frequency of, e.g.,between about 1 minute and about 6 hours (e.g., between about 1 minuteand about 5 hours, between about 1 minute and about 4 hours, betweenabout 1 minute and about 3 hours, between about 1 minute and 2 hours,between about 1 minute and 1 hour, or between about 1 minute and 30minutes), depending on, e.g., the chromatography column(s) and/orchromatographic membrane(s) used in the MCCS or the MCCS1 and MCCS2.

Culturing Methods

Some of the processes described herein further include a step ofculturing cells (e.g., recombinant mammalian cells) that secrete arecombinant protein in a bioreactor (e.g., a perfusion or fed-batchbioreactor) that includes a liquid culture medium, wherein a volume ofthe liquid culture medium that is substantially free of cells (e.g.,mammalian cells) is continuously or periodically removed from thebioreactor (e.g., perfusion bioreactor) and fed into the MCCS or MCCS1.The bioreactor can have a volume of, e.g., between about 1 L to about10,000 L (e.g., between about 1 L to about 50 L, between about 50 L toabout 500 L, between about 500 L to about 1000 L, between 500 L to about5000L, between about 500 L to about 10,000 L, between about 5000 L toabout 10,000 L, between about 1 L and about 10,000 L, between about 1Land about 8,000 L, between about 1 L and about 6,000 L, between about 1L and about 5,000 L, between about 100 L and about 5,000 L, betweenabout 10 L and about 100 L, between about 10 L and about 4,000 L,between about 10 L and about 3,000 L, between about 10 L and about 2,000L, or between about 10 L and about 1,000 L). The amount of liquidculture medium present in a bioreactor can be, e.g., between aboutbetween about 0.5 L to about 5,000 L (e.g., between about 0.5 L to about25 L, between about 25 L to about 250 L, between about 250 L to about500 L, between 250 L to about 2500 L, between about 250 L to about 5,000L, between about 2500 L to about 5,000 L, between about 0.5 L and about5,000 L, between about 0.5 L and about 4,000 L, between about 0.5 L andabout 3,000 L, between about 0.5 L and about 2,500 L, between about 50 Land about 2,500 L, between about 5 L and about 50 L, between about 5 Land about 2,000 L, between about 5 L and about 1,500 L, between about 5L and about 1,000 L, or between about 5 L and about 500 L). Culturingcells can be performed, e.g., using a fed-batch bioreactor or aperfusion bioreactor. Non-limiting examples and different aspects ofculturing cells (e.g., culturing mammalian cells) are described belowand can be used in any combination.

Cells

The cells that are cultured in some of the processes described hereincan be bacteria (e.g., gram negative bacteria), yeast (e.g.,Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, orArxula adeninivorans), or mammalian cells. The mammalian cell can be acell that grows in suspension or an adherent cell. Non-limiting examplesof mammalian cells that can be cultured in any of the processesdescribed herein include: Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS/0),B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells(e.g., HEK 293E and HEK 293F), African green monkey kidney epithelialcells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidneyepithelial cells (MDCK) cells. In some examples where an adherent cellis cultured, the culture can also include a plurality of microcarriers(e.g., microcarriers that include one or more pores). Additionalmammalian cells that can be cultured in any of the processes describedherein are known in the art.

The mammalian cell can include a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant protein (e.g., a recombinant protein).Non-limiting examples of recombinant nucleic acids that encode exemplaryrecombinant proteins are described below, as are recombinant proteinsthat can be produced using the methods described herein. In someinstances, the mammalian cell that is cultured in a bioreactor (e.g.,any of the bioreactors described herein) was derived from a largerculture.

A nucleic acid encoding a recombinant protein can be introduced into amammalian cell using a wide variety of methods known in molecularbiology and molecular genetics. Non-limiting examples includetransfection (e.g., lipofection), transduction (e.g., lentivirus,adenovirus, or retrovirus infection), and electroporation. In someinstances, the nucleic acid that encodes a recombinant protein is notstably integrated into a chromosome of the mammalian cell (transienttransfection), while in others the nucleic acid is integrated.Alternatively or in addition, the nucleic acid encoding a recombinantprotein can be present in a plasmid and/or in a mammalian artificialchromosome (e.g., a human artificial chromosome). Alternatively or inaddition, the nucleic acid can be introduced into the cell using a viralvector (e.g., a lentivirus, retrovirus, or adenovirus vector). Thenucleic acid can be operably linked to a promoter sequence (e.g., astrong promoter, such as a β-actin promoter and CMV promoter, or aninducible promoter). A vector including the nucleic acid can, ifdesired, also include a selectable marker (e.g., a gene that confershygromycin, puromycin, or neomycin resistance to the mammalian cell).

In some instances, the recombinant protein is a secreted protein and isreleased by the mammalian cell into the extracellular medium (e.g., thefirst and/or second liquid culture medium). For example, a nucleic acidsequence encoding a soluble recombinant protein can include a sequencethat encodes a secretion signal peptide at the N- or C-terminus of therecombinant protein, which is cleaved by an enzyme present in themammalian cell, and subsequently released into the extracellular medium(e.g., the first and/or second liquid culture medium).

Culture Media

Liquid culture media are known in the art. The liquid culture media(e.g., a first and/or second tissue culture medium) can be supplementedwith a mammalian serum (e.g., fetal calf serum and bovine serum), and/ora growth hormone or growth factor (e.g., insulin, transferrin, andepidermal growth factor). Alternatively or in addition, the liquidculture media (e.g., a first and/or second liquid culture medium) can bea chemically-defined liquid culture medium, an animal-derived componentfree liquid culture medium, a serum-free liquid culture medium, or aserum-containing liquid culture medium. Non-limiting examples ofchemically-defined liquid culture media, animal-derived component freeliquid culture media, serum-free liquid culture media, andserum-containing liquid culture media are commercially available.

A liquid culture medium typically includes an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The liquid culture media (e.g., a first and/or second liquidculture medium) can, if desired, be supplemented with, e.g., a mammalianhormone or growth factor (e.g., insulin, transferrin, or epidermalgrowth factor), salts and buffers (e.g., calcium, magnesium, andphosphate salts), nucleosides and bases (e.g., adenosine, thymidine, andhypoxanthine), protein and tissue hydrolysates, and/or any combinationof these additives.

A wide variety of different liquid culture media that can be used toculture cells (e.g., mammalian cells) in any of the methods describedherein are known in the art. Medium components that also may be usefulin the present processes include, but are not limited to,chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides(two or more amino acids), and CD growth factors. Additional examples ofliquid tissue culture medium and medium components are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different media.

Additional Features of Exemplary Bioreactors

The interior surface of any of the bioreactors described herein may haveat least one coating (e.g., at least one coating of gelatin, collagen,poly-L-ornithine, polystyrene, and laminin), and as is known in the art,one or more ports for the sparging of O₂, CO₂, and N₂ into the liquidculture medium, and a stir mechanism for agitating the liquid culturemedium. The bioreactor can incubate the cell culture in a controlledhumidified atmosphere (e.g., at a humidity of greater than 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidity of 100%).The bioreactor can also be equipped with a mechanical device that iscapable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., anATF system or the cell filtering system described in U.S. ProvisionalPatent Application Ser. No. 61/878,502).

Temperature

The step of culturing of mammalian cells can be performed at atemperature of about 31° C. to about 40° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in during the culturing step, e.g., on an hourly or daily basis. Forexample, the temperature can be changed or shifted (e.g., increased ordecreased) at about one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, fourteen days, fifteen days, sixteen days, seventeen days,eighteen days, nineteen days, or about twenty days or more after theinitial seeding of the bioreactor with the cell (e.g., mammalian cell).For example, the temperature can be shifted upwards (e.g., a change ofup to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20°C.). For example, the temperature can be shifted downwards (e.g., achange of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to orabout 20° C.).

CO₂

The culturing step described herein can further include exposing theliquid culture medium in the bioreactor to an atmosphere including atmost or about 15% CO₂ (e.g., at most or about 14% CO₂, 12% CO₂, 10% CO₂,8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂, 3% CO₂, 2% CO₂, or at most or about 1%CO₂).

Perfusion Bioreactor

The culturing step described herein can be performed using a perfusionbioreactor. Culturing a cell (e.g., a mammalian cell) in a perfusionbioreactor includes the removal from the bioreactor of a first volume ofa first liquid culture medium (e.g., including any concentration ofmammalian cells, e.g., a first volume of a first liquid culture mediumthat is substantially free of cells), and adding to the first liquidculture medium a second volume of a second liquid culture medium.Removal and adding can be performed simultaneously or sequentially, or acombination of the two. Further, removal and adding can be performedcontinuously (e.g., at a rate that removes and replaces a volume ofbetween 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%,between 1% and 500%, between 1% and 400%, between 1% and 350%, between1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, or between 4% and 30%) of the volume of thebioreactor or the first liquid culture medium volume over any given timeperiod (e.g., over a 24-hour period, over an incremental time period ofabout 1 hour to about 24 hours, or over an incremental time period ofgreater than 24 hours)) or periodically (e.g., once every third day,once every other day, once a day, twice a day, three times a day, fourtimes a day, or five times a day), or any combination thereof. Whereperformed periodically, the volume that is removed or replaced (e.g.,within about a 24-hour period, within an incremental time period ofabout 1 hour to about 24 hours, or within an incremental time period ofgreater than 24 hours) can be, e.g., between 0.1% to 800% (e.g., between1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and400%, between 1% and 300%, between 1% and 200%, between 1% and 100%,between 100% and 200%, between 5% and 150%, between 10% and 50%, between15% and 40%, between 8% and 80%, or between 4% and 30%) of the volume ofthe bioreactor or the first liquid culture medium volume. The firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added can in some instances be heldapproximately the same over each 24-hour period (or, alternatively, anincremental time period of about 1 hour to about 24 hours or anincremental time period of greater than 24 hours) over the entire orpart of the culturing period. As is known in the art, the rate at whichthe first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., by a mechanical system that can remove the first volume of thefirst liquid culture medium from the bioreactor (e.g., the first volumeof the first liquid culture medium that is substantially free of cellsfrom the bioreactor). Alternatively or in addition, the first volume ofthe first liquid culture medium can be removed by seeping or gravityflow of the first volume of the first liquid culture medium through asterile membrane with a molecular weight cut-off that excludes the cell(e.g., mammalian cell).

The second volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Fed-Batch Bioreactor

The culturing step described herein can be performed using a fed-batchbioreactor. Culturing a cell in a fed-batch bioreactor includes, overthe majority of the culturing period, the addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. The adding of the secondliquid culture medium can be performed continuously (e.g., at a ratethat adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume over any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, or between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different. The volume of the secondliquid culture medium can be added to the first liquid culture medium inan automated fashion, e.g., by perfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur within atleast 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours,within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9hours, within 10 hours, within 12 hours, within 14 hours, within 16hours, within 18 hours, within 24 hours, within 36 hours, within 48hours, within 72 hours, within 96 hours, or after 96 hours) of theseeding of the bioreactor with a mammalian cell. The cell culture mediumin fed-batch cultures is typically harvested at the end of cultureperiod and used in any of the processes described herein, however, thecell culture medium in fed-batch cultures can also be harvested at oneor more time points during the culturing period and used in any of theprocesses described herein.

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., containers, volumes, rates or frequencies of replacingculture volumes, agitation frequencies, temperatures, media, and CO₂concentrations) can be used in any combination in to perform thesemethods. Further, any of the mammalian cells described herein or knownin the art can be used to produce a recombinant protein.

Exemplary Biological Manufacturing Systems

Examples of biological manufacturing systems useful for performing theprocesses described herein and that include a MCCS or a MCCS1 and MCCS2are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390 (incorporated by reference). In theseexemplary systems, at least one (e.g., at least two, three, four, five,or six) at least one reduced bioburden packed chromatography columnprovided herein is present in the MCCS or in the MCCS1 and/or MCCS2. Forexample, the entire system can include a total of two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of thereduced bioburden packed chromatography columns provided herein. Forexample, the MCCS, MCCS1, and/or MCCS2 can include (or can each include)one, two, three, four, five, six, seven, eight, nine, or ten of thereduced bioburden packed chromatography columns provided herein.

For example, useful systems can include a MCCS1 that includes an inletand a MCCS2 that includes an outlet, or an MCCS that includes an inletand an outlet. In some embodiments, the MCCS1 and MCCS2 are in fluidcommunication with each other. These systems can also be configured suchthat fluid can be passed into the inlet, through the MCCS1 and MCCS2,and exit the manufacturing system through the outlet. These systemsprovide for the continuous and time-efficient production of atherapeutic drug substance from a liquid culture medium. For example,the elapsed time between feeding a fluid (e.g., a liquid culture medium)including a therapeutic protein into the MCCS1 and eluting purifiedrecombinant protein (e.g., therapeutic protein drug substance) from theoutlet of the MCCS2 can be, e.g., between about 4 hours and about 48hours, inclusive.

Some exemplary systems do not include a break tank. In others, thesystem can include a maximum of 1, 2, 3, 4, or 5 break tank(s) in theentire system (e.g., where each break tank only holds a therapeuticprotein for a total time period of, e.g., between about 5 minutes andabout 6 hours, inclusive). The break tank(s) can have a capacity that isbetween 1 mL and about 300 mL, inclusive. Any break tank(s) disposed inthe system such that fluid enters the break tank(s) prior to enteringMCCS1 or MCCS can have a capacity that is between 1 mL and about 100%,inclusive, of the loading volume of the first column of the MCCS1 orMCCS, respectively. Any break tanks(s) disposed in the system such thatfluid enters the break tank(s) prior to entering the MCCS2 (and afterexiting the MCCS1) can have a capacity that is, e.g., between 1 mL andabout 100%, inclusive, of the loading volume of the first column of theMCCS2.

Additional Exemplary System Structures and Features

The MCCS or MCCS1 can include an inlet through which fluid (e.g., aliquid culture medium that is substantially free of cells) can be passedinto the MCCS or MCCS1, respectively. The inlet can be any structureknown in the art for such purposes. It can include, e.g., a threading,ribbing, or a seal that allows for a fluid conduit to be inserted, suchthat after insertion of the fluid conduit into the inlet, fluid willenter the MCCS or MCCS1 through the inlet without significant seepage offluid out of the inlet. Non-limiting inlets that can be used in thepresent systems are known and would be understood by those in the art.

The MCCS or MCCS1 can include at least two chromatography columns, atleast two chromatographic membranes, or at least one chromatographycolumn and at least one chromatographic membrane, and an inlet. The MCCSor MCCS1 can be any of the exemplary MCCSs described herein, or have oneor more of any of the exemplary features of an MCCS (in any combination)described herein. The chromatography column(s) and/or thechromatographic membrane(s) present in the MCCS or MCCS1 can have one ormore of any of the exemplary shapes, sizes, volumes (bed volumes),and/or unit operation(s) described herein.

The chromatography column(s) and/or the chromatographic membrane(s)present in the MCCS or MCCS1 can include one or more of any of theexemplary resins described herein or known in the art. For example, theresin included in one or more of the chromatography column(s) and/orchromatographic membrane(s) present in the MCCS or MCCS1 can be a resinthat utilizes a capture mechanism (e.g., protein A-binding capturemechanism, protein G-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, substrate-binding capture mechanism,cofactor-binding capture mechanism, an aptamer-binding capturemechanism, and/or a tag-binding capture mechanism). The resin includedin one or more of the chromatography column(s) and/or chromatographicmembrane(s) of the MCCS or MCCS1 can be a cation exchange resin, ananion exchange resin, a molecular sieve resin, or a hydrophobicinteraction resin, or any combination thereof. Additional examples ofresins that can be used to purify a recombinant protein are known in theart, and can be included in one or more of the chromatography column(s)and/or chromatographic membrane(s) present in the MCCS or MCCS1. Thechromatography column(s) and/or chromatography membranes present in theMCCS or MCCS1 can include the same and/or different resins (e.g., any ofthe resins described herein or known in the art for use in recombinantprotein purification).

The two or more chromatography column(s) and/or chromatographic resin(s)present in the MCCS or MCCS1 can perform one or more unit operations(e.g., capturing a recombinant protein, purifying a recombinant protein,polishing a recombinant protein, inactivating viruses, adjusting theionic concentration and/or pH of a fluid including the recombinantprotein, or filtering a fluid including a recombinant protein). Innon-limiting examples, the MCCS or MCCS1 can perform the unit operationsof capturing a recombinant protein from a fluid (e.g., a liquid culturemedium) and inactivating viruses present in the fluid including therecombinant protein. The MCCS or MCCS1 can perform any combinations oftwo of more unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS or MCCS1 can be connected or moved with respect to eachother by a switching mechanism (e.g., a column-switching mechanism). TheMCCS or MCCS1 can also include one or more (e.g., two, three, four, orfive) pumps (e.g., automated, e.g., automated peristaltic pumps). Thecolumn-switching events can be triggered by the detection of a level ofrecombinant protein detected by UV absorbance corresponding to a certainlevel of recombinant protein in the fluid passing through the MCCS orMCCS1 (e.g., the input into and/or eluate from one or more of thechromatography column(s) and/or chromatographic membranes in the MCCS orMCCS1), a specific volume of liquid (e.g., buffer), or specific timeelapsed. Column switching generally means a mechanism by which at leasttwo different chromatography columns and/or chromatographic membranes inan MCCS or MCCS1 (e.g., two or more different chromatography columnsand/or chromatographic membranes present in the MCCS1 or MCCS2) areallowed to pass through a different step (e.g., equilibration, loading,eluting, or washing) at substantially the same time during at least partof the process.

The MCCS or MCCS1 can be a Periodic Counter-Current Chromatographysystem (PCCS). For example, the PCCS that is the MCCS or MCCS1 (i.e.,PCCS or PCCS1, respectively) can include four chromatography columns,where the first three columns perform the unit operation of capturing arecombinant protein from a fluid (e.g., a liquid culture medium), andthe fourth column of the PCCS performs the unit operation ofinactivating viruses in the fluid including the recombinant protein. APCCS that is the MCCS or MCCS1 can utilize a column-switching mechanism.The PCC system can utilize a modified ÄKTA system (GE Healthcare,Piscataway, N.J.) capable of running up to, e.g., four, five, six,seven, or eight columns, or more.

The MCCS or MCCS1 can be equipped with: one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV monitors, one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) valves,one or more (e.g., two, three, four, five, six, seven, eight, nine, orten) pH meters, and/or one or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) conductivity meters. The MCCS or MCCS1 canalso be equipped with an operating system that utilizes software (e.g.,Unicorn-based software, GE Healthcare, Piscataway, N.J.) for sensingwhen a column-switching should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting (triggering) thecolumn-switching events.

The MCCS or MCCS1 can further include one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty,twenty-one, twenty-two, twenty-three, or twenty-four) in-line bufferadjustment reservoir(s) and/or a buffer reservoir(s). In other examples,the MCCS or MCCS1 can include one or more (e.g., two, three, four, five,or six) break tanks that can hold fluid that cannot readily pass intoone or more of the chromatography columns and/or chromatographicmembranes in the MCCS or MCCS1. The systems described herein can includeone or more break tanks (e.g., a break tank described herein) in theMCCS, MCCS1, and/or MCCS2. Other examples of the systems describedherein do not include a break tank in the MCCS, MCCS1, or MCCS2, or donot include a break tank in the entire system. Other examples of thesystems described herein include a maximum of one, two, three, four, orfive break tank(s) (e.g., any break tank(s) described herein) in theentire system.

Second MCCS

The second MCCS (MCCS2) in the exemplary systems includes at least twochromatography columns, at least two chromatographic membranes, or atleast one chromatography column(s) and at least one chromatographicmembrane(s), and an outlet. The MCCS2 can any of the exemplary MCCSsdescribed herein, or can have one or more of any of the exemplaryfeatures of an MCCS (in any combination) described herein. Thechromatography column(s) and/or the chromatographic membrane(s) presentin the MCCS2 can have one or more of: any of the shapes, sizes, volumes(bed volumes), and/or unit operations described herein. Thechromatography column(s) and/or the chromatographic membrane(s) caninclude any of the exemplary resins described herein or known in theart. For example, the resin included in one or more of thechromatography column(s) and/or chromatographic membrane(s) present inthe MCCS2 can be a resin that utilizes a capture mechanism (e.g.,protein A-binding capture mechanism, protein G-binding capturemechanism, antibody- or antibody fragment-binding capture mechanism,substrate-binding capture mechanism, cofactor-binding capture mechanism,tag-binding capture mechanism, and/or aptamer-binding capturemechanism). Useful resins include, e.g., a cation exchange resin, ananion exchange resin, a molecular sieve resin, and a hydrophobicinteraction resin. Additional examples of resins are known in the art.The chromatography column(s) and/or chromatography membranes present inthe MCCS2 can include the same and/or different resins (e.g., any of theresins described herein or known in the art for use in recombinantprotein purification).

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can perform one or more unit operations (e.g., any of theunit operations described herein or any combination of the unitoperations described herein). In non-limiting examples, the MCCS2 canperform the unit operations of purifying a recombinant protein from afluid and polishing the recombinant protein present in the fluidincluding the recombinant protein. In other non-limiting examples, theMCCS2 can perform the unit operations of purifying a recombinant proteinpresent in a fluid, polishing a recombinant protein present in a fluid,and filtering a fluid including a recombinant protein. In anotherexample, the MCCS2 can perform the unit operations of purifying arecombinant protein present in a fluid, polishing a recombinant proteinpresent in a fluid, filtering a fluid including a recombinant protein,and adjusting the ionic concentration and/or pH of a fluid including arecombinant protein. The MCCS2 can perform any combination of two ofmore unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can be connected or moved with respect to each other by aswitching mechanism (e.g., a column-switching mechanism). The MCCS2 canalso include one or more (e.g., two, three, four, or five) pumps (e.g.,automated, e.g., automated peristaltic pumps). The column-switchingevents can be triggered by the detection of a level of recombinantprotein detected by UV absorbance corresponding to a certain level ofrecombinant protein in the fluid passing through the MCCS2 (e.g., theinput into and/or eluate from one or more of the chromatographycolumn(s) and/or chromatographic membranes in the MCCS2), a specificvolume of liquid (e.g., buffer), or specific time elapsed.

The MCCS2 can be a Periodic Counter-Current Chromatography system (i.e.,PCCS2). For example, the PCCS2 can include three columns that performthe unit operation of purifying a recombinant protein from a fluid, anda chromatographic membrane that performs the unit operation of polishinga recombinant protein present in a fluid. For example, the three columnsthat perform the unit operation of purifying a recombinant protein froma fluid can include, e.g., a cationic exchange resin, and thechromatographic membrane that performs the unit operation of polishingcan include a cationic exchange resin. A PCCS2 can utilize acolumn-switching mechanism. The PCCS2 can utilize a modified ÄKTA system(GE Healthcare, Piscataway, N.J.) capable of running up to, e.g., four,five, six, seven, or eight columns, or more.

The MCCS2 can be equipped with: one or more (e.g., two, three, four,five, six, seven, eight, nine, or ten) UV monitors, one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) valves, one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten) pHmeters, and/or one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) conductivity meters. The MCCS2 can also be equippedwith an operating system that utilizes software (e.g., Unicorn-basedsoftware, GE Healthcare, Piscataway, N.J.) for sensing when acolumn-switching event should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting the column-switchingevents.

The MCCS2 can further include one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three, or twenty-four) in-line buffer adjustmentreservoir(s) and/or a buffer reservoir(s). In other examples, the MCCS2can include one or more (e.g., two, three, four, five, or six) breaktanks (e.g., any of the break tanks described herein) that can holdfluid that cannot readily pass into one or more of the chromatographycolumns and/or chromatographic membranes in the MCCS2.

The MCCS2 includes an outlet through which the therapeutic protein drugsubstance can exit the system. The outlet can include, e.g., athreading, ribbing, or a seal that allows for a fluid conduit to beinserted or a vial designed to hold or store the purified recombinantprotein (e.g., therapeutic protein drug substance). An outlet caninclude a surface that can be used to seal a reduced bioburden vial orother such storage container onto the outlet in order to allow thepurified recombinant protein (e.g., therapeutic protein drug substance)to flow directly into the reduced bioburden vial or storage container.Non-limiting outlets that can be used in the present systems are knownand would be understood by those in the art.

The systems described herein can also include a fluid conduit that isdisposed between the MCCS1 and the MCCS2. Any of the fluid conduitsdescribed herein can be, e.g., a tube that is made of, e.g.,polyethylene, polycarbonate, or plastic. The fluid conduit disposedbetween the MCCS1 and the MCCS2 can further include one of more of thefollowing in any combination: one or more in-line buffer adjustmentreservoirs that are in fluid communication with the fluid conduit andare positioned such that the buffer stored within the in-line bufferadjustment reservoir(s) is added to the fluid present in the fluidconduit; a break tank (e.g., any of the break tank(s) described herein)that is in fluid communication with the fluid conduit and is positionedsuch that it can hold any excess fluid present in the fluid conduit thatis unable to readily feed into the MCCS2; and one or more filters thatare disposed in the fluid conduit such that they are capable offiltering (e.g., removing bacteria) the fluid present in the fluidconduit. Any of the in-line buffer adjustment reservoirs can include,e.g., a volume of between about 0.5 L to 50 L of buffer (e.g., at atemperature at or below 25° C., 15° C., or 10° C.).

The systems described herein can optionally include a fluid conduitdisposed between the final chromatography column or chromatographicmembrane in the MCCS2 and the outlet. The systems described herein canfurther include one or more filters in fluid connection with the fluidconduit disposed between the final chromatography column orchromatographic membrane in the MCCS2 and the outlet, such that thefilter can remove, e.g., precipitated material, particulate matter, orbacteria from the fluid present in the fluid conduit disposed betweenthe final chromatography column or chromatographic membrane in the MCCS2and the outlet.

Some examples of the systems provided herein also include a bioreactorthat is in fluid connectivity with the inlet of the MCCS or MCCS1. Anyof the exemplary bioreactors described herein or known in the art can beused in the present systems.

Some examples of the systems provided herein also include a pump system.A pump system can include one or more the following: one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) pumps (e.g.,any of the pumps described herein or known in the art), one or more(e.g., two, three, four, or five) filters (e.g., any of the filtersdescribed herein or known in the art), one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV detectors, and one ormore (e.g., two, three, four, or five) break tanks (e.g., any of thebreak tanks described herein). Some examples of the systems providedherein further include a fluid conduit disposed between the pump and theinlet of the MCCS or MCCS1 (e.g., any of the exemplary fluid conduitsdescribed herein or known in the art). In some examples, this particularfluid conduit can include one or more (e.g., two, three, or four) pumps(e.g., any of the pumps described herein or known in the art) and/or oneor more (e.g., two, three, or four) break tanks (e.g., any of theexemplary break tanks described herein), where these pump(s) and/orbreak tank(s) are in fluid connection with the fluid present in thefluid conduit.

Some examples of the systems described herein further include a furtherfluid conduit connected to the fluid conduit between the pump and theinlet, where one end of the further fluid conduit is fluidly connectedto a bioreactor and the other end is fluidly connected to the fluidconduit between the pump and the inlet. This further fluid conduit caninclude a filter that is capable of removing cells from the liquidculture medium removed from the bioreactor (e.g., ATF cell retentionsystem).

The systems provided herein allow for the continuous production of apurified recombinant protein (e.g., therapeutic protein drug substance).As is known in the art, the systems can provide for the periodic elutionof a purified recombinant protein (e.g., therapeutic protein drugsubstance). The systems described herein can also result in a net yieldof purified recombinant protein (e.g., therapeutic protein drugsubstance) of at least about 5 g/day, at least about 10 g/day, at leastabout 15 g/day, at least about 20 g/day, at least about 30 g/day, or atleast about 40 g/day over a continuous period of at least about 5 days,at least about 10 days, at least about 15 days, at least about 20 days,at least about 25 days, at least about 30 days, at least about 40 days,at least about 50 days, at least about 60 days, at least about 70 days,at least about 80 days, at least about 90 days, or least about 100 days.

Methods of Reducing Bioburden of a Chromatography Resin that Include theUse of a Substantially Dry Chromatography Resin

Also provided herein are methods of reducing bioburden of achromatography resin that include (a) exposing a container comprising asubstantially dry chromatography resin to a dose of gamma-irradiationsufficient to reduce the bioburden of the container and thechromatography resin, wherein the substantially dry chromatography resinincludes a liquid including at least one alcohol, where the at least onealcohol is present in an amount sufficient to ameliorate the loss ofbinding capacity of the chromatography resin after exposure to the doseof gamma-irradiation. Some embodiments of these methods further includeprior to step (a) drying a chromatography resin to substantially removeliquid (but not all liquid) from the chromatography resin. Drying of achromatography resin can be performed using heat treatment (e.g., anoven) or a dessicator. Additional methods for drying a chromatographyresin are known in the art.

Any of the conditions and doses for gamma-irradiation described hereincan be used in these methods. For example, the dose of gamma-irradiationcan be between about 15 kGy to about 45 kGy (e.g., between about 20 kGyto about 30 kGy). Any of the containers, chromatography resins, andliquids including at least one alcohol described herein can be used inthese methods. For example, the container can be a storage vessel or achromatography column. The chromatography resin in these methods caninclude a protein ligand (e.g., protein A or protein G). In someexamples, the chromatography resin can include an anionic exchangechromatography resin (e.g., a chromatography resin includingN-benzyl-N-methyl-ethanolamine groups). In some examples, thechromatography resin is covalently attached to a surface of an article(e.g., a chip, membrane, or cassette). In some embodiments, thesubstantially dry chromatography resin does not contain a significantamount of an antioxidant agent or a significant amount of a chelator.Also provided are reduced bioburden chromatography resins produced byany of the methods described herein.

In some examples, the reduced bioburden chromatography resin producedhas a sterility assurance level (SAL) of between about 1×10⁻⁸ to about1×10⁻⁵ (e.g., a SAL of between about 1×10⁻⁷ to about 1×10⁻⁶). Thereduced chromatography resin produced can include at least one resinselected from the group consisting of: anionic exchange chromatographyresin, cationic exchange chromatography resin, affinity chromatographyresin, hydrophobic interaction chromatography resin, and size exclusionchromatography resin. In some examples, the reduced chromatography resinproduced includes an affinity chromatography resin comprising a proteinligand (e.g., protein A). In some examples, the reduced chromatographyresin produced includes an anionic exchange chromatography resin (e.g.,an anionic exchange chromatography resin includingN-benzyl-N-methyl-ethanolamine groups). Also provided are methods ofmaking a reduced bioburden packed chromatography column that includeproviding the reduced bioburden chromatography resin produced by any ofthe methods described herein; and packing the chromatography resin intoa reduced bioburden column in an aseptic environment. Also provided arereduced bioburden packed chromatography columns produced by any of themethods described herein.

Also provided are integrated, closed, and continuous processes forreduced bioburden manufacturing of a purified recombinant protein thatinclude: (a) providing a liquid culture medium including a recombinantprotein that is substantially free of cells; and (b) continuouslyfeeding the liquid culture medium into a multi-column chromatographysystem (MCCS) comprising at least one reduced bioburden packedchromatography column produced by any of the methods provided herein;where the process utilizes reduced bioburden buffer, is integrated, andruns continuously from the liquid culture medium to an eluate from theMCCS that is the purified recombinant protein. Also provided areintegrated, closed, and continuous processes for reduced bioburdenmanufacturing of a purified recombinant protein that include: (a)providing a liquid culture medium including a recombinant protein thatis substantially free of cells; (b) continuously feeding the liquidculture medium into a first multi-column chromatography system (MCCS1);(c) capturing the recombinant protein in the liquid culture medium usingthe MCCS1; (d) producing an eluate from the MCCS1 that includes therecombinant protein and continuously feeding the eluate into a secondmulti-column chromatography system (MCCS2); (e) continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, where: the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to the purified recombinant protein, and at least one column inthe MCCS1 and/or MCCS2 contains a reduced bioburden packedchromatography column produced by any of the methods provided herein.Any of the exemplary aspects of integrated, closed, and continuousprocesses for reduced bioburden manufacturing of a purified recombinantprotein described herein can be used in these processes.

Methods of Producing Reduced Bioburden Membranes, Resins, CoatedMaterials, Chips, and Cassettes

Also provided herein are methods for producing a reduced bioburdenmembrane, resin, coated material, chip, or cassette that include:exposing a container including a composition including (i) a membrane,resin, coated material, chip, or cassette (e.g., a cellulose-, agarose-,or a sugar-based membrane, resin, coated material, chip, or cassette);and (ii) a liquid including at least one alcohol (e.g., and optionally,at least one antioxidant agent and/or chelator) to a dose ofgamma-irradiation sufficient to reduce the bioburden of the containerand the membrane, resin, coated material, chip, or cassette, where theat least one alcohol is present in an amount sufficient to amelioratethe damage to the membrane, resin, coated material, chip, and cassetteafter exposure to the dose of gamma-irradiation. In some examples, thecassette is a resin-containing cassette (e.g., any of the exemplaryresins described herein or known in the art). In some embodiments, themembrane, resin, coated material, chip, or cassette includes a protein Aor protein G ligand covalently attached to at least one or part of itssurface. In some embodiments, the composition includes a membrane,resin, coated material, chip, or cassette and the liquid including theat least alcohol (e.g., and optionally, at least one antioxidant agentand/or at least one chelator). Any of the exemplary combinations andconcentrations of alcohols, antioxidant agent(s), and/or chelator(s)described herein can be used in any of these methods. Any of theexemplary liquids described herein can be used in any of these methods.In some embodiments, the composition is a wetted or moist dry material.Also provided herein is a reduced bioburden membrane, resin, coatedmaterial, chip, or cassette produced using any of the methods describedherein. In some examples the container is a sealed storage container orvessel.

Also provided herein are methods for producing a reduced bioburdenmembrane, resin, coated material, chip, and cassette that include:exposing a container including a substantially dry membrane, resin,coated material, chip, or cassette (e.g., a cellulose-, agarose-, or asugar-based membrane, resin, coated material, chip, or cassette) to adose of gamma-irradiation sufficient to reduce the bioburden of thecontainer and the membrane, resin, coated material, chip, or cassette.Some examples further include a step of drying the membrane, resin,coated materials, chip, or cassette prior to the exposing step. In someembodiments, the membrane, resin, coated material, chip, or cassetteincludes a protein A or protein G ligand covalently attached to itssurface. In some examples, the cassette is a resin-containing cassette(e.g., any of the exemplary resins described herein or known in theart). Also provided herein is a reduced bioburden membrane, resin,coated material, chip, or cassette produced using any of the methodsdescribed herein.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Protective Effect of an Alcohol on a Gamma-IrradiatedAffinity Chromatography Resin

In a first set of experiments, MabSelect™ SuRe™ (Protein A affinitychromatography resin) was irradiated in three different buffers at adose of 40-49 kGy with a standard dosing rate:

(1) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mMmannitol in 50 mM sodium phosphate buffer (40-49 kGy irradiation at adose rate of >7.5 kGy/hour);

(2) 2% v/v benzyl alcohol (40-49 kGy at a dose rate of >7.5 kGy/hour);and

(3) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mMMannitol, 2% benzyl alcohol in 50 mM sodium phosphate buffer—at higherirradiation dose (40-49 kGy and higher dose rate of >7.5 kGy/hour)

Following irradiation, the chromatography resins were packed intoseparate chromatography columns and cycled using a cell culture harvestand a residence time of six minutes. The breakthrough binding capacitieswere recorded for irradiated resins, and were compared to naïve(non-irradiated) MabSelect™ SuRe™ (Protein A chromatography) resin.

The data shown for this experiment are shown in FIG. 1.

The data in FIG. 1 show that buffer (3) provides an additive effect ofthe combination of both buffers (1) and (2) to retain binding capacity.In this instance, the 2% v/v benzyl alcohol in the buffer works as apreservative, and when combined with buffer (1), also offers someprotective properties.

Table 1 shows that all of the product quality attributes measured didnot very significantly based on the use of buffers (1)-(3), and aresimilar to the expected values seen with naïve (non-irradiated)MabSelect™ SuRe™ (Protein A chromatography resin).

TABLE 1 Quality attributes Recovery HCP Residual ProA Buffer Cyclenumber (%) (ppm) (ppm) Naïve MSS 1 92 233 17 5 89 304 16 10 90 174 34Buffer 3 1 89 245 27 SMM′H + 2% BA 5 87 233 25 10 88 225 26 Buffer 1 189 274 34 SMM′H 5 93 273 37 10 94 245 25 Buffer 2 1 89 310 26 2% BA 5 87294 27 10 88 287 25

This data show that irradiation of a chromatography resin in a liquidincluding an alcohol (e.g., benzyl alcohol) protects the resin againstsubsequent loss in binding capacity over multiple cycles ofchromatography.

Example 2 Protective Effect of an Alcohol on a Gamma-Irradiated Capto™Adhere Chromatography Resin

Gamma irradiation of GE Capto™ adhere (anion exchange chromatographyresin with multimodal functionality) was irradiated in three differentbuffers at a dose of 28-49 kGy with a standard dosing rate:

(A) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, and 25 mMmannitol in 50 mM sodium phosphate buffer (28-34 kGy at dose rate of 2-3kGy/hour);

(B) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mMmannitol in 50 mM sodium phosphate buffer (40-49 kGy at a dose rateof >7.5 kGy/hour); and

(C) 25 mM sodium ascorbate, 25 mM methionine, 25 mM histidine, 25 mMmannitol, 2% v/v benzyl alcohol in 50 mM sodium phosphate buffer (40-49kGy and higher dose rate of >7.5 kGy/hour).

Following irradiation, the chromatography resins were packed intoseparate chromatography columns and cycled using a cell culture harvestand a residence time of six minutes. The breakthrough binding capacitieswere recorded for irradiated resins, and were compared to naïve(non-irradiated) GE Capto™ adhere (anion exchange chromatography resinwith multimodal functionality).

The data from this experiment are shown in FIG. 2. The data in FIG. 2show that no appreciable change in binding capacity is observed when theresin is irradiated in the presence of benzyl alcohol, and the presenceof benzyl alcohol provides the benefit of increased storage time priorto gamma irradiation.

Table 2 below shows that irradiation of the resin in the presence ofbenzyl alcohol did not lead to any appreciable impact on other qualityattributes of the resin's performance in protein purification.

TABLE 2 Quality attributes Cycle HCP Buffer number (μg/mg) Purity (%)Test Condition A 1 229 92.32 SMM′H low dose 10 279 90.64 (28-34 kGy) 18218 91.31 Test Condition B 1 285 92.01 SMM′H high dose 10 162 90.58(40-49 kGy) 18 207 90.82 Test Condition C 2 165 91.69 SMM′H + 2% BA 11166 91.8 (40-49 kGy) 16 221 92.28

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of reducing bioburden of a chromatography resin comprising:exposing a container comprising a composition comprising (i) achromatography resin and (i) a liquid comprising at least one alcohol,to a dose of gamma-irradiation sufficient to reduce the bioburden of thecontainer and the chromatography resin, wherein the at least one alcoholis present in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin after exposure to the dose ofgamma-irradiation.
 2. (canceled)
 3. The method of claim 1, wherein thecontainer is a storage vessel.
 4. The method of claim 1, wherein thecontainer is a chromatography column.
 5. The method of claim 1, whereinthe container is a packed chromatography column.
 6. The method of claim1, wherein the composition is a slurry of sedimented chromatographyresin.
 7. (canceled)
 8. The method of claim 1, wherein the at least onealcohol is selected from the group of: benzyl alcohol, cyclohexanol,isobutyl alcohol, 2-methyl-2-butanol, methanol, ethanol, propan-2-ol,propan-1-ol, butan-1-ol, pentan-1-ol, hexadecan-1-ol, 2-phenyl ethanol,see-phenyl ethanol, 3-phenyl-1-propanol, 1-phenyl-1-propanol,2-phenyl-1-propanol, 2-phenyl-2-propanol, 1-phenyl-2-butanol,2-phenyl-1--butanol, 3-phenyl-1-butanol, 4-phenyl-2-butanol,dl-1-phenyl-2-pentanol, 5-phenyl-1-pentanol, and 4-phenyl-1-butanol. 9.The method of claim 8, wherein the at least one alcohol comprises benzylalcohol.
 10. The method of claim 1, wherein the total sum concentrationof the one or more alcohols in the liquid is about 0.01% v/v to about10% v/v.
 11. The method of claim 1, wherein the liquid further comprisesat least one antioxidant agent and/or chelator.
 12. The method of claim11, wherein the liquid comprises at least one antioxidant agent and/orchelator in an amount sufficient to ameliorate the loss of bindingcapacity of the chromatography resin after exposure to the dose ofgamma-irradiation.
 13. The method of claim 12, wherein the liquidcomprises at least one antioxidant agent selected from the groupconsisting of: reduced glutathione, reduced thioredoxin, reducedcysteine, a carotenoid, melatonin, lycopene, tocopherol, reducedubiquinone, ascorbate, bilirubin, uric acid, lipoic acid, a flavonoid, aphenolpropanoid acid, lidocaine, naringenin, fullerene, glucose,mannitol, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, anddimethylmethoxy chromanol.
 14. The method of claim 13, wherein theliquid comprises at least one antioxidant agent selected from the groupconsisting of: mannitol, sodium ascorbate, histidine, and methionine.15. The method of claim 14, wherein the liquid comprises mannitol,sodium ascorbate, histidine, and methionine.
 16. The method of claim 12,wherein the liquid comprises: (i) between 75 mM and about 125 mMmannitol; (ii) between 75 mM and about 125 mM methionine; (iii) between75 mM and about 125 mM sodium ascorbate; (iv) between 75 mM and about125 mM histidine; (v) between 30 mM and about 70 mM methionine andbetween about 30 mM and about 70 mM histidine; (vi) between about 10 mMand about 50 mM methionine, between about 10 mM and about 50 mMhistidine, and between about 10 mM and about 50 mM sodium ascorbate; or(vii) between about 5 mM to about 45 mM sodium ascorbate, between about5 mM and about 45 mM methionine, between about 5 mM and about 45 mMmannitol, and between about 5 mM to about 45 mM histidine. 17.(canceled)
 18. The method of claim 12, wherein the liquid comprises atleast one chelator selected from the group consisting of:ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanesulfonic acid sodium (DMPS), dimercaptosuccinicacid (DMSA), metallothionin, and desferroxamine.
 19. The method of claim1, wherein the chromatography resin is selected from the groupconsisting of: anionic exchange chromatography resin, cationic exchangechromatography resin, affinity chromatography resin, hydrophobicinteraction chromatography resin, and size exclusion chromatographyresin.
 20. The method of claim 19, wherein the composition comprisesaffinity chromatography resin comprising a protein ligand.
 21. Themethod of claim 20, wherein the protein ligand is protein A.
 22. Themethod of claim 19, wherein the composition comprises an anionicexchange chromatography resin.
 23. (canceled)
 24. The method of claim 1,wherein the dose is between about 15 kGy to about 45 kGy. 25.-28.(canceled)
 29. A reduced bioburden chromatography resin produced by themethod of claim
 3. 30.-36. (canceled)
 37. A method of making a reducedbioburden packed chromatography column comprising: providing the reducedbioburden chromatography resin of claim 29; and packing thechromatography resin into a reduced bioburden column in an asepticenvironment.
 38. A reduced bioburden packed chromatography columnproduced by the method of claim
 37. 39. A reduced bioburden packedchromatography column produced by the method of claim
 5. 40.-46.(canceled)
 47. A composition comprising (i) a chromatography resin and(ii) a liquid comprising at least one alcohol, wherein the at least onealcohol is present in an amount sufficient to ameliorate the loss ofbinding capacity of the chromatography resin upon treatment with a doseof gamma-irradiation sufficient to reduce bioburden of the composition.48.-63. (canceled)
 64. A method of performing reduced bioburden columnchromatography comprising: (a) providing a reduced bioburden packedchromatography column of claim 38; and (b) performing columnchromatography using the reduced bioburden packed chromatography columnand reduced bioburden buffer in a closed system. 65.-74. (canceled) 75.An integrated, closed, and continuous process for reduced bioburdenmanufacturing of a purified recombinant protein comprising: (a)providing a liquid culture medium comprising a recombinant protein thatis substantially free of cells; and (b) continuously feeding the liquidculture medium into a multi-column chromatography system (MCCS)comprising at least one reduced bioburden packed chromatography columnof claim 38; wherein the process utilizes reduced bioburden buffer, isintegrated, and runs continuously from the liquid culture medium to aneluate from the MCCS that is the purified recombinant protein. 76.-79.(canceled)
 80. The process of claim 75, wherein the MCCS comprises atleast two reduced bioburden packed chromatography columns.
 81. Theprocess of claim 75, wherein the MCCS is a periodic counter currentchromatography system.
 82. The process of claim 75, wherein the MCCSincludes a plurality of columns for affinity or pseudo-affinitychromatography, cation exchange chromatography, anion exchangechromatography, or size exclusion chromatography, or any combinationthereof. 83.-84. (canceled)
 85. An integrated, closed, and continuousprocess for reduced bioburden manufacturing of a purified recombinantprotein comprising: (a) providing a liquid culture medium comprising arecombinant protein that is substantially free of cells; (b)continuously feeding the liquid culture medium into a first multi-columnchromatography system (MCCS1); (c) capturing the recombinant protein inthe liquid culture medium using the MCCS1; (d) producing an eluate fromthe MCCS1 that comprises the recombinant protein and continuouslyfeeding the eluate into a second multi-column chromatography system(MCCS2); (e) continuously feeding the recombinant protein from theeluate into the MCCS2 and subsequently eluting the recombinant proteinto thereby produce the purified recombinant protein, wherein: theprocess utilizes reduced bioburden buffer, is integrated, and runscontinuously from the liquid culture medium to the purified recombinantprotein, and at least one column in the MCCS1 and/or MCCS2 contains areduced bioburden packed chromatography column of claim
 38. 86.-102.(canceled)